M-CSF-Specific Monoclonal Antibody and Uses Thereof

ABSTRACT

M-CSF-specific RX1-based or RX-I derived antibodies are provided, along with pharmaceutical compositions containing such antibody, kits containing a pharmaceutical composition, and methods of preventing and treating bone loss in or remodeling in a subject afflicted with an osteoblastic or osteolytic disease.

TECHNICAL FIELD

This invention relates to methods for preventing and treating,osteolysis, cancer metastasis and bone loss or remodeling associatedwith cancer metastasis by administering an M-CSF-specific antibody to asubject.

BACKGROUND OF THE INVENTION

Cancer metastasis is the primary cause of post-operation or post-therapyrecurrence in cancer patients. Despite intensive efforts to developtreatments, cancer metastasis remains substantially refractory totherapy. Bone is one of the most common sites of metastasis of varioustypes of human cancers (e.g., breast, lung, prostate and thyroidcancers). The occurrence of osteolytic bone metastases causes seriousmorbidity due to intractable pain, high susceptibility to fracture,nerve compression and hypercalcemia. Despite the importance of theseclinical problems, there are few available treatments for cancermetastasis or bone remodeling associated with cancer metastasis.

The balance between the activities of osteoblasts (bone-forming cells)and osteoclasts (bone-lysing cells) in general determines the phenotypeof metastatic bone lesions, and both osteoblasts and osteoclasts havebeen implicated—directly or indirectly—in bone metastasis. Bonemetastases with a relative bone-forming (osteoblastic) phenotype are theresult of comparative stimulation of osteoblasts or inhibition ofosteoclasts (or both) by the cancer cells, whereas metastases with arelative bone-lysing (osteolytic) phenotype reflect comparativeinhibition of osteoblasts or stimulation of osteoclast function (orboth) by the cancer cells. Metastases from prostate cancer, most ofwhich are adenocarcinomas, nearly always form lesions in bone that aremore osteoblastic than osteolytic; by contrast, bone metastases fromkidney, lung or breast cancers more often are osteolytic. However,metastases from the relatively uncommon neuroendocrine tumours of theprostate also produce osteolytic lesions. Additionally, patients canexhibit a mixed form of disease with osteoblastic and lytic lesionspresent.

Osteoclasts mediate bone readsorption osteoclasts are multinucleatedcells differentiated from haemopoietic cells. It is generally acceptedthat osteoclasts are formed by the fusion of mononuclear precursorsderived from haemopoietic stem cells in the bone marrow, rather thanincomplete cell divisions (Chambers, Bone and Mineral Research, 6: 1-25,1989; Göthling et al., Clin Orthop Relat R. 120: 201-228, 1976; Kahn etal., Nature 258: 325-327, 1975, Suda et al., Endocr Rev 13: 66-80, 1992;Walker, Science 180: 875, 1973; Walker, Science 190: 785-787, 1975;Walker, Science 190: 784-785, 1975). They share a common stem cell withmonocyte-macrophage lineage cells (Ash et al., Nature 283: 669-670,1980, Kerby et al., J. Bone Miner Res 7: 353-62, 1992). Thedifferentiation of osteoclast precursors into mature multinucleatedosteoclasts requires different factors including hormonal and localstimuli (Athanasou et al., Bone Miner 3: 317-333, 1988; Feldman et al.,Endocrinology 107: 1137-1143, 1980; Walker, Science 190: 784-785, 1975;Zheng et al., Histochem J 23: 180-188, 1991) and living bone and bonecells have been shown to play a critical role in osteoclast development(Hagenaars et al., Bone Miner 6: 179-189, 1989). Osteoblastic or bonemarrow stromal cells are also required for osteoclast differentiation.One of the factors produced by these cells that supports osteoclastformation is macrophage-colony stimulating factor, M-CSF(Wiktor-Jedrzejczak et al., Proc Natl Acad Sci USA 87: 4828-4832, 1990;Yoshida et al., Nature 345: 442-444, 1990). Receptor activator for NF-κBligand (RANKL, also known as TRANCE, ODF and OPGL) is another signal(Suda et al., Endocr Rev 13: 66-80, 1992) through whichosteoblastic/stromal cells stimulate osteoclast formation and resorptionvia a receptor, RANK (TRANCER), located on osteoclasts and osteoclastprecursors (Lacey et al., Cell 93: 165-176, 1998; Tsuda et al., BiochemBiophys Res Co 234: 137-142, 1997; Wong et al., J Exp Med 186:2075-2080, 1997; Wong et al., J Biol. Chem 272: 25190-25194, 1997;Yasuda et al., Endocrinology 139: 1329-1337, 1998; Yasuda et al., ProcNatl Acad Sci US 95: 3597-3602, 1998). Osteoblasts also secrete aprotein that strongly inhibits osteoclast formation calledosteoprotegerin (OPG, also known as OCIF), which acts as a decoyreceptor for the RANKL thus inhibiting the positive signal betweenosteoclasts and osteoblasts via RANK and RANKL.

Osteoclasts are responsible for dissolving both the mineral and organicbone matrix (Blair et al., J Cell Biol 102: 1164-1172, 1986).Osteoclasts represent terminally differentiated cells expressing aunique polarized morphology with specialized membrane areas and severalmembrane and cytoplasmic markers, such as tartrate resistant acidphosphatase (TRAP) (Anderson et al. 1979), carbonic anhydrase II(Väänänen et al., Histochemistry 78: 481-485, 1983), calcitonin receptor(Warshafsky et al., Bone 6: 179-185, 1985) and vitronectin receptor(Davies et al., J Cell Biol 109: 1817-1826, 1989). Multinucleatedosteoclasts usually contain less than 10 nuclei, but they may contain upto 100 nuclei being between 10 and 100 μm in diameter (Göthling et al.,Clin Orthop Relat R 120: 201-228, 1976). This makes them relatively easyto identify by light microscopy. They are highly vacuolated when in theactive state, and also contain many mitochondria, indicative of a highmetabolic rate (Mundy, in Primer on the metabolic bone diseases anddisorders of mineral metabolism, pages 18-22, 1990). Since osteoclastsplay a major role in osteolytic bone metastases, as well as otherosteolytic diseases such as osteoporosis, for example, there is a needin the art for new agents and methods for preventing osteoclaststimulation and function.

Thus, there remains a need in the art to identify new agents and methodsfor preventing or treating osteolysis or cancer metastasis, includingmixed osteoblastic and osteolytic bone metastases and bone diseasesassociated with increased osteoclast activity.

SUMMARY OF THE INVENTION

The materials and methods of the present invention fulfill theaforementioned and other related needs in the art. In one embodiment ofthe invention, a non-murine monoclonal antibody is provided, includingfunctional fragment, that specifically binds to the same epitope ofM-CSF as any one of murine monoclonal antibody RX1, MC1, or MC3 havingthe amino acid sequences set forth in FIGS. 4, 14, and 15, respectively.In a related embodiment, an aformentioned antibody is provided whereinthe antibody is selected from the group consisting of a polyclonalantibody; a monoclonal antibody including a Human Engineered™ antibody;a humanized antibody; a human antibody; a chimeric antibody; Fab,F(ab′)₂; Fv; Sc Fv or SCA antibody fragment; a diabody; linear antibody;or a mutein of any one of these antibodies, that preferably retainbinding affinity of at least 10⁻⁷, 10⁻⁸ or 10⁻⁹ or higher. A non-murinemonoclonal antibody, including functional fragment, that competes withmonoclonal antibody RX1, MC1, and/or MC3 having the amino acid sequenceset forth in FIG. 4 for binding to M-CSF by more than 75%, is alsocontemplated.

In another embodiment, a non-murine monoclonal antibody, includingfunctional fragment, wherein said non-murine monoclonal antibody orfunctional fragment thereof binds an epitope of M-CSF that includes atleast 4, 5, 6, 7 or 8 contiguous residues of amino acids 98-105 of FIG.12 is provided.

In another embodiment, the invention provides a non-murine monoclonalantibody, including functional fragment, wherein said non-murinemonoclonal antibody or functional fragment thereof binds an epitope ofM-CSF that includes at least 4, 5, 6, 7 or 8 contiguous residues ofamino acids 65-73 or 138-144 of FIG. 12 (corresponding to M-CSF epitopesrecognized by 5H4 or MC3).

In yet another embodiment, the aforementioned antibody or fragment thatbinds an epitope of M-CSF that includes amino acids 98-105 of FIG. 12 isprovided. In a related embodiment, the aforementioned antibody isprovided comprising CDR3 of FIG. 4A. In another embodiment, the antibodyis provided comprising at least 1, 2, 3, 4, 5, or 6 CDRs of murineantibody RX1 set forth in FIG. 4A. Such an antibody that comprises atleast 1, 2, 3, 4, or 5 CDRs of murine antibody RX1 may also comprise atleast 1, 2, 3, 4, or 5 CDRs of any of the 6 CDRs of antibody 5H4 setforth in FIG. 16A-B. Alternatively, the antibody that comprises at least1, 2, 3, 4, or 5 CDRs of murine antibody RX1 may also comprise at least1, 2, 3, 4, or 5 CDRs of any of the 6 CDRs of antibody MC1 set forth inFIG. 16A-B. In yet another alternative, the aforementioned antibody mayalso comprise at least 1, 2, 3, 4, or 5 CDRs of any of the 6 CDRs ofantibody MC3 set forth in FIG. 16A-B. In a related embodiment, theantibody that comprises at least 1, 2, 3, 4, or 5 CDRs of murineantibody RX1 may comprise at least 1, 2, 3, 4 or 5 CDRs of the consensusCDRs set forth in FIG. 16A-B is provided. In still another relatedembodiment, in the aforementioned antibody one or more residues of theconsensus CDR(s) is substituted by the corresponding residue of any ofthe CDRs of antibody murine RX1, 5H4, MC1 or MC3. The desired bindingaffinity may be retained even though one or more of the amino acids inthe antibody have been mutated, e.g. by conservative substitutions inthe CDRs, and/or conservative or non-conservative changes in the low andmoderate risk residues.

In another embodiment of the invention, variants of the aforementionedantibody are provided, comprising a variable heavy chain amino acidsequence which is at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, or 99% homologous to the amino acid sequence set forthin FIG. 4A, 13, 14, or 15. In a related embodiment, the antibodycomprises a variable light chain amino acid sequence which is at least60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%homologous to the amino acid sequence set forth in FIG. 4A, 13, 14, or15.

In yet another embodiment, the antibody comprises a constant region andone or more heavy and light chain variable framework regions of a humanantibody sequence. In a related embodiment, the antibody comprises amodified or unmodified constant region of a human IgG1, IgG2, IgG3 orIgG4. In a preferred embodiment, the constant region is human IgG1 orIgG4, which may optionally be modified to enhance or decrease certainproperties. In the case of IgG1, modifications to the constant region,particularly the hinge or CH2 region, may increase or decrease effectorfunction, including ADCC and/or CDC activity. In other embodiments, anIgG2 constant region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constant region,particularly the hinge region, may reduce the formation ofhalf-antibodies.

In yet another embodiment of the invention, the aforementioned antibodyis derived from, based on, or contains part of a human consensussequence, human germline sequence, human consensus germline sequence, orany one of the human antibody sequences in Kabat, NCBI Ig Blast,http://www.ncbi.nlm.nih.gov/igblast/showGermline.cgi, Kabat Databasehttp://www.bioinf.org.uk/abs/seqtest.html, FTP site for Kabat Release5.0 (1992) ftp://ftp.ncbi.nih.gov/repository/kabat/Rel5.0/,ImMunoGeneTics database (Montpellier France) http://imgt.cnusc.fr:8104/,V-Base http://www.mrc-cpe.cam.ac.uk/LIST.php?menu=901, Zurich Universityhttp://www.unizh.ch/antibody/Sequences/index.html, The TherapeuticAntibody Human Homology Project (TAHHP)http://www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html, ProteinSequence and Structure Analysis of Antibody Domainshttp://how.to/AnalyseAntibodies/, Humanization by designhttp://people.cryst.bbk.ac.uk/˜ubcg07s/, Antibody Resourceshttp://www.antibodyresource.com/educational.html, Antibody Engineering(by TT Wu), Humana Press.

In a preferred aspect of the invention, the aforementioned antibody is aHuman Engineered™ antibody. For example, the Human Engineered™ antibodysequence is any one of the sequences set forth in FIGS. 23-24. OtherHuman Engineered™ antibodies or variants thereof are contemplated.

For example, in one embodiment, the aforementioned RX1-based antibody isprovided, wherein the heavy chain variable region comprises the aminoacid sequenceX₁VX₂LX₃EX₄GX₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃LX₁₄LX₁₅CX₁₆VX₁₇DYSITSDYAWNWIX₁₈QX₁₉X₂₀X₂₁X₂₂X₂₃LX₂₄WMGY1SYSGSTSX₂₅NX₂₆X₂₇LX₂₈X₂₉X₃₀IX₃₁IX₃₂RX₃₃X₃₄X₃₅X₃₆X₃₇X₃₈FX₃₉LX₄₀LX₄₁X₄₂VX₄₃X₄₄X₄₅DX₄₆AX₄₇YYCASFDYAHAMDYWGX₄₈GTX₄₉VX₅₀VX₅₁X₅₂(SEQ ID NO: 124), wherein X is any amino acid. In a related embodiment,the antibody is provided wherein the heavy chain variable regioncomprises the amino acid sequenceDVX₁LX₂EX₃GPX₄X₅VX₆PX₇X₈X₉LX₁₀LX₁₁CX₁₂VTDYSITSDYAWNWIRQX₁₃PX₁₄X₁₅KLEWMGYISYSGSTSYNPSLKX₁₆RIX₁₇IX₁₈RX₁₉TX₂₀X₂₁NX₂₂FX₂₃LX₂₄LX₂₅X₂₆VX₂₇X₂₈X₂₉DX₃₀ATYYCASFDYAHAMDYWGX₃₁GTX₃₂VX₃₃VX₃₄X₃₅(SEQ ID NO: 125), wherein X is any amino acid.

In still another embodiment of the invention, the aforementionedantibody is provided, wherein the heavy chain variable region comprisesthe amino acid sequenceX₁VQLQESGPGLVKPSQX₂LSLTCTVX₃DYSITSDYAWNWIRQFPGX₄X₅LEWMGYISYSGSTSYNPSLKSRIX₆IX₇RDTSKNQFX₈LQLNSVTX₉X₁₀DTAX₁₁YYCASFDYAHAMDYWGQGTX₁₂VTVSS (SEQ ID NO: 126), wherein X is any amino acid. In arelated embodiment, the antibody is provided, wherein the heavy chainvariable region comprises the amino acid sequenceDVQLQESGPGLVKPSQX₁LSLTCTVTDYSITSDYAWNWIRQFPGX₂KLEWMGYISYSGSTSYNPSLKSRIX₃IX₄RDTSKNQFX₅LQLNSVTX₆X₇DTATYYCASFDYAHAMDYWGQ GTX₈VTVSS(SEQ ID NO: 127), wherein X is any amino acid. In yet anotherembodiment, the antibody is provided wherein the heavy chain variableregion comprises the amino acid sequenceDVQLQESGPGLVKPSQTLSLTCTVTDYSITSDYAWNWIRQFPGKKLEWMGYISYSGSTSYNPSLKSRITISRDTSKNQFSLQLNSVTAADTATYYCASFDYAHAMDYWGQGTTV TVSS (SEQ IDNO: 41). In still another embodiment, the antibody is provided whereinthe heavy chain variable region comprises the amino acid sequence

(SEQ ID NO: 43) QVQLQESGPGLVKPSQTLSLTCTVSDYSITSDYAWNWIRQFPGKGLEWMGYISYSGSTSYNPSLKSRITISRDTSKNQFSLQLNSVTAADTAVYYCASFD YAHAMDYWGQGTTVTVSS.

In another embodiment of the invention, the aforementioned antibody isprovided wherein the light chain variable region comprises the aminoacid sequenceX₁IX₂LX₃QX₄X₅X₆X₇X₈X₉VX₁₀X₁₁X₁₂X₁₃X₁₄VX₁₅FX₁₆CX₁₇AX₁₈QSIGTSIHWYX₁₉QX₂₀X₂₁X₂₂X₂₃X₂₄PX₂₅LLIKYASEX₂₆X₂₇X₂₈X₂₉IX₃₀X₃₁X₃₂FX₃₃GX₃₄GX₃₅GX₃₆X₃₇FX₃₈LX₃₉LX₄₀X₄₁VX₄₂X₄₃X₄₄DX₄₅ADYYCQQINSWPTTFGX₄₆GTX₄₇LX₄₈X₄₉X₅₀X₅₁X₅₂(SEQ ID NO: 128), wherein X is any amino acid. In a related embodiment,the antibody is provided wherein the light chain variable regioncomprises the amino acid sequenceX₁IX₂LX₃QX₄PX₅X₆LX₇VX₈PX₉X₁₀X₁₁VX₁₂FX₁₃CX₁₄ASQSIGTSIHWYQQX₁₅TX₁₆X₁₇SPRLLIKYASEX₁₈ISX₁₉IPX₂₀RFX₂₁GX₂₂GX₂₃GX₂₄X₂₅FX₂₆LX₂₇IX₂₈X₂₉VX₃₀X₃₁X₃₂DX₃₃ADYYCQQINSWPTTFGX₃₄GTX₃₅LX₃₆X₃₇X₃₈X₃₉X₄₀ (SEQ ID NO: 129), wherein X isany amino acid. In yet another embodiment, the antibody is providedwherein the light chain variable region comprises the amino acidsequenceX₁IX₂LTQSPX₃X₄LSVSPGERVX₅FSCRASQSIGTSIHWYQQX₆TX₇X₈X₉PRLLIKYASEX₁₀X₁₁X₁₂GIPX₁₃RFSGSGSGTDFTLX₁₄IX₁₅X₁₆VESEDX₁₇ADYYCQQINSWPTTFGX₁₈GTKLEIKRX₁₉ (SEQ ID NO: 130), wherein X is any amino acid.

In another embodiment of the invention, the aforementioned antibody isprovided wherein the light chain variable region comprises the aminoacid sequenceX₁IX₂LTQSPX₃X₄LSVSPGERVX₅FSCRASQSIGTSIHWYQQX₆TX₇X₈SPRLLIKYASEX₉ISGIPX₁₀RFSGSGSGTDFTLX₁₁IX₁₂X₁₃VESEDX₁₄ADYYCQQINSWPTTFGX₁₅GTKLEIK RX₁₆(SEQ ID NO: 131), wherein X is any amino acid. In a related embodiment,the antibody is provided wherein the light chain variable regioncomprises the amino acid sequenceX₁IX₂LTQSPX₃X₄LSVSPGERVX₅FSCRASQSIGTSIHWYQQX₆TX₇X₈X₉PRLLIKYASESISGIPX₁₀RFSGSGSGTDFTLX₁₁IX₁₂X₁₃VESEDX₁₄ADYYCQQINSWPTTFGX₁₅GTKLEIK RX₁₆(SEQ ID NO: 132), wherein X is any amino acid. In yet anotherembodiment, the antibody is provided wherein the light chain variableregion comprises the amino acid sequence

(SEQ ID NO: 45) EIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQAPRLLIKYASESISGIPDRFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQ GTKLEIKRT.

In still another embodiment of the invention, the aforementionedantibody is provided wherein the light chain variable region comprisesthe amino acid sequenceEIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQAPRLLIKYASERATGIPDRFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQGTKLEIKRT (SEQ ID NO: 47). Inanother embodiment, the antibody is provided, wherein the light chainvariable region comprises the amino acid sequence

(SEQ ID NO: 48) EIVLTQSPGTLSVSPGERVTFSCRASQSIGTSIHWYQQKTGQSPRLLIKYASERISGIPDRFSGSGSGTDFTLTISRVESEDFADYYCQQINSWPTTFGQ GTKLEIKRT.

In another embodiment of the invention, the aforementioned antibodywherein at least one X is a corresponding amino acid within the aminoacid sequence set forth in FIG. 4A is provided. In a related embodiment,the antibody is provided wherein at least one X is a conservativesubstitution (according to Table 1) of a corresponding amino acid withinthe amino acid sequence set forth in FIG. 4A. In yet another relatedembodiment, the antibody is provided wherein at least one X is anon-conservative substitution (according to Table 1) of a correspondingamino acid within the amino acid sequence set forth in FIG. 4A. In stillanother embodiment of the invention, the antibody is provided wherein atleast one X is a corresponding amino acid within a human antibodysequence.

The aforementioned Human Engineered™ antibody is derived from, based on,or contains part of the human antibody consensus sequence, humangermline sequence, human consensus germline sequence, or any one of thehuman antibody sequences in Kabat, NCBI Ig Blast,http://www.ncbi.nlm.nih.gov/igblast/showGermline.cgi, Kabat Databasehttp://wwvv.bioinforg.uldabs/seqtest.html, FTP site for Kabat Release5.0 (1992) ftp://ftp.ncbi.nih.gov/repository/kabat/Rel5.0/,ImMunoGeneTics database (Montpellier France) http://imgt.cnusc.fr:8104/,V-Base http://wvvw.mrc-cpe.cam.ac.uk/LIST.php?menu=901, ZurichUniversity http://www.unizh.ch/˜antibody/Sequences/index.html, TheTherapeutic Antibody Human Homology Project (TAHHP)http://www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html, ProteinSequence and Structure Analysis of Antibody Domainshttp://how.to/AnalyseAntibodies/, Humanization by designhttp://people.cryst.bbk.ac.uk/˜ubcg07s/, Antibody Resourceshttp://www.antibodyresource.com/educational.html, Antibody Engineering(by TT Wu), Humana Press.

In another embodiment of the invention, the aforementioned antibodywherein the Human Engineered™ antibody sequence is one of the sequencesset forth in FIG. 23-24 or 29-30 is provided. In another embodiment, theantibody comprises a variable heavy chain amino acid sequence which isat least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identical to one of the heavy chain amino acid sequences set forthin FIG. 19B. In yet another embodiment, the antibody comprises avariable light chain amino acid sequence which is at least 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to oneof the light chain amino acid sequences set forth in any of FIGS.20B-22B.

In yet another embodiment, the aforementioned antibody such as 5H4, MC1or MC3 antibody with the sequences set forth in one of FIGS. 24C-24E isHuman Engineered™ according to the methods set forth in Studnicka etal., U.S. Pat. No. 5,766,886 and Example 5 herein, using the Kabatnumbering set forth in FIGS. 24C-24E to identify low, moderate and highrisk residues. In a related embodiment, the aforementioned antibody isprovided wherein all of the low risk residues of either the heavy orlight chain or both are modified, where necessary, to be the sameresidues as a human reference immunoglobulin sequence. Similarly,another embodiment of the invention provides the aforementioned antibodywherein all of the low+moderate risk residues of either the heavy orlight chain or both are modified, where necessary, to be the sameresidues as a human reference immunoglobulin sequence. A heavy chainwherein all of the low risk residues have been modified may be combinedwith a light chain wherein all of the low and moderate risk residueshave been modified, and vice versa. Similarly, an aforementioned HumanEngineered™ light or heavy chain May be combined with a light or heavychain of a humanized or chimeric antibody.

In still another embodiment, the antibody comprises a variable heavychain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to one of the heavychain amino acid sequences described immediately above Human Engineered™according to the Studnicka method. In yet another embodiment, theantibody comprises a variable light chain amino acid sequence which isat least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% identical to one of the light chain amino acid sequences describedimmediately above Human Engineered™ according to the Studnicka method.

In still another embodiment, an antibody is provided comprising a heavychain as set forth above and a light chain as set forth above.

In yet another embodiment of the invention, the aforementioned antibodyhas an affinity Kd of at least 10[−7]. In a related embodiment, theantibody has an affinity Kd of at least 10[9−9].

In another embodiment of the invention, the aforementioned antibody isprovided which is a polyclonal antibody; a monoclonal antibody includinga Human Engineered™ antibody; a humanized antibody; a human antibody; achimeric antibody; Fab, F(ab′)₂, Fv, ScFv or SCA antibody fragment; adiabody; a linear antibody; or a mutein of any one of these antibodies.In a related embodiment, the monoclonal antibody is an isolatedantibody.

In still another embodiment of the invention, an isolated nucleic acidis provided comprising a nucleic acid sequence encoding a light chain ofthe aforementioned antibody. In a related embodiment, the isolatednucleic acid comprises a heavy chain nucleic acid sequence which is atleast 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%identical to the heavy chain nucleotide sequence set forth in FIG. 4A,13, 14, or 15. In yet another related embodiment, the isolated nucleicacid comprises a light chain nucleic acid sequence which is at least 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identicalto the light chain nucleotide sequence set forth in FIG. 4A, 13, 14, or15.

In another embodiment, a vector comprising the aforementioned isolatednucleic acid is provided. In a related embodiment, the aforementionedvector is provided wherein the isolated nucleic acid is operably linkedto a regulatory control sequence. In still another embodiment, a hostcell is provided comprising the aforementioned vector.

Numerous methods are contemplated in the present invention. For example,a method of producing an aforementioned antibody is provided comprisingculturing the aforementioend host cell such that the isolated nucleicacid is expressed to produce the antibody. In a related embodiment, themethod further comprises the step of recovering the antibody from thehost cell culture. In a related embodiment, an isolated antibodyproduced by the aforementioned method is provided.

A hybridoma that secretes an aforementioned antibody is also provided bythe present invention. Additionally, an aforementioned antibody that isconjugated to a toxin is provided.

In another embodiment of the invention, a pharmaceutical composition isprovided comprising any one of the aforementioned antibodies and apharmaceutically suitable carrier, excipient or diluent. In a relatedembodiment, the pharmaceutical composition further comprises a secondtherapeutic agent. In yet another related embodiment, the pharmaceuticalcomposition is provided wherein the second therapeutic agent is a cancerchemotherapeutic agent. In still another related embodiment, thepharmaceutical composition is provided wherein the second therapeuticagent is a bisphosphonate. In another embodiment the second therapeuticagent is another antibody.

Antibodies of the present invention are contemplated to have numerousdesirable characteristics for the treatment of diseases and disorders.In one embodiment of the invention, the any one of aforementionedantibodies that bind to M-CSF for preventing a subject afflicted with adisease that causes or contributes to or results from-osteolysis,wherein the antibody effectively reduces the severity of bone loss orremodeling associated with the disease, is provided. Similarly, any oneof aforementioned antibodies that bind to M-CSF is provided for treatinga subject afflicted with a disease that causes or contributes to orresults from osteolysis, wherein said antibody effectively reduces theseverity of bone loss or remodeling associated with the disease.

Numerous diseases and disorders are contemplated to be amenable toantibody-based treatment in the present invention. In one embodiment ofthe invention, the aforementioned antibody is provided wherein saiddisease is selected from the group consisting of osteolytic diseasesassociated with relatively increased osteoclast activity or theosteoclastic/osteoblastic process required for bone remodeling(associated with both increased osteoclastic activity and increasedosteoblastic activity), including metabolic bone diseases,endocrinopathies (hypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, hyperthyroidism), hypercalcemia, deficiency states(rickets/osteomalacia, scurvy, malnutrition), chronic diseases(malabsorption syndromes, chronic renal failure (renal osteodystrophy),chronic liver disease (hepatic osteodystrophy)), drugs (glucocorticoids(glucocorticoid-induced osteoporosis), heparin, alcohol), and hereditarydiseases (osteogenesis imperfecta, homocystinuria), cancer,osteoporosis, periprosthetic bone loss (for example, due to a foreignimplant in bone or joint replacement with a prosthesis, including totalhip arthroplasty), -inflammation of bone associated with arthritis andrheumatoid arthritis, periodontal disease, fibrous dysplasia, and/orPaget's disease.

In a related embodiment, the aforementioned antibody that binds to M-CSFis provided for preventing or treating metastatic cancer to bone,wherein the metastatic cancer is breast, lung, renal, multiple myeloma,thyroid, prostate, adenocarcinoma, blood cell malignancies, includingleukemia and lymphoma; head and neck cancers; gastrointestinal cancers,including esophageal cancer, stomach cancer, colon cancer, intestinalcancer, colorectal cancer, rectal cancer, pancreatic cancer, livercancer, cancer of the bile duct or gall bladder; malignancies of thefemale genital tract, including ovarian carcinoma, uterine endometrialcancers, vaginal cancer, and cervical cancer; bladder cancer; braincancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer,including malignant melanoma or squamous cell cancer.

In another embodiment of the invention, a method of screening for anM-CSF-specific antibody is provided comprising the steps of contactingmetastatic tumor cell medium, osteoclasts and a candidate antibody;detecting osteoclast formation, proliferation and/or differentiation;and identifying said candidate antibody as a M-CSF-specific antibody ifa decrease in osteoclast formation, proliferation and/or differentiationis detected. Similarly, the aforementioned method is provided whereinsaid metastatic tumor cell medium includes tumor cells.

In a related embodiment, the aforementioned method is provided furthercomprising the step of determining if the candidate antibody binds toM-CSF. Similarly, another embodiment of the invention provides theaforementioned method further comprising the step of determining if saidcandidate antibody inhibits interaction between M-CSF and its receptorM-CSFR.

In another embodiment of the invention, a method of identifying anM-CSF-specific antibody that can prevent or treat metastatic cancer tobone is provided, comprising the steps of: (a) detecting binding of acandidate antibody to an epitope of M-CSF that includes at least 4contiguous residues of amino acids 98-105 of FIG. 12; and (b) assayingthe ability of said candidate antibody to prevent or treat metastaticcancer to bone in vitro or in vivo.

In another embodiment of the invention, a method of identifying anM-CSF-specific antibody that can prevent or treat metastatic cancer tobone is provided, comprising the steps of: (a) detecting binding of acandidate antibody to an epitope of M-CSF that includes at least 4contiguous residues of amino acids 65-73 or 138-144 of FIG. 12(corresponding to M-CSF epitopes recognized by 5H4 or MC3); and (b)assaying the ability of said candidate antibody to prevent or treatmetastatic cancer to bone in vitro or in vivo.

In yet another embodiment of the invention, a method of altering a CDRof an antibody that binds an epitope of M-CSF that includes amino acids98-105 of FIG. 12 is provided comprising altering an amino acid within aCDR of the amino acid sequence set forth in FIG. 4A and selecting for anantibody that binds M-CSF with an affinity Ka of at least 10[−7]. Inanother embodiment, a method of systematically altering up to 60% of theheavy chain amino acid sequence set forth in FIG. 4A is providedcomprising altering any of X1-X52 in the amino acid sequenceX₁VX₂LX₃EX₄GX₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃LX₁₄LX₁₅CX₁₆VX₁₇DYSITSDYAWNWIX₁₈QX₁₉X₂₀X₂₁X₂₂X₂₃LX₂₄WMGYISYSGSTSX₂₅NX₂₆X₂₇LX₂₈X₂₉X₃₀IX₃₁IX₃₂RX₃₃X₃₄X₃₅X₃₆X₃₇X₃₈FX₃₉LX₄₀LX₄₁X₄₂VX₄₃X₄₄X₄₅DX₄₆AX₄₇YYCASFDYAHAMDYWGX₄₈GTX₄₉VX₅₀VX₅₁X₅₂(SEQ ID NO: 133), and testing an antibody comprising the altered aminoacid sequence for binding to an epitope of M-CSF that includes aminoacids 98-105 of FIG. 12.

In a related embodiment, a method of systematically altering up to 60%of the light chain amino acid sequence set forth in FIG. 4A is providedcomprising altering any of X1-X52 in the amino acid sequenceX₁IX₂LX₃QX₄X₅X₆X₇X₈X₉VX₁₀X₁₁X₁₂X₁₃X₁₄VX₁₅FX₁₆CX₁₇AX₁₈QSIGTSIHWYX₁₉QX₂₀X₂₁X₂₂X₂₃X₂₄PX₂₅LLIKYASEX₂₆X₂₇X₂₈X₂₉IX₃₀X₃₁X₃₂FX₃₃GX₃₄GX₃₅GX₃₆X₃₇FX₃₈LX₃₉IX₄₀X₄₁VX₄₂X₄₃X₄₄DX₄₅ADYYCQQINSWMFGX₄₆GTX₄₇LX₄₈X₄₉X₅₀X₅₁X₅₂(SEQ ID NO: 134), and testing an antibody comprising the altered aminoacid sequence for binding to an epitope of M-CSF that includes aminoacids 98-105 of FIG. 12.

In yet another embodiment of the invention, a method of altering a CDRof an antibody that binds an epitope of M-CSF that includes amino acids65-73 or 138-144 of FIG. 12 (corresponding to M-CSF epitopes recognizedby 5H4 or MC3) is provided comprising altering an amino acid within aCDR of the amino acid sequence set forth in one of FIGS. 13, 14, and 15,and selecting for an antibody that binds M-CSF with an affinity Ka of atleast 10[−7]. In another embodiment, a method of systematically alteringup to 60% of the heavy chain amino acid sequence set forth in one ofFIGS. 13, 14, and 15, is provided comprising altering the aforementionedsequences according to the methods set forth in Studnicka et al., U.S.Pat. No. 5,766,886 and Example 5 herein, and according to the Kabatnumbering set forth in FIGS. 24C-24E, and testing an antibody comprisingthe altered amino acid sequence for binding to an epitope of M-CSF thatincludes amino acids 65-73 or 138-144 of FIG. 12 (corresponding to M-CSFepitopes recognized by 5H4 or MC3). In a related embodiment, all of thelow risk residues are modified. Similarly, in another embodiment of theinvention all of the low and moderate risk residues are modified. In yetanother embodiment, all of the low and moderate risk residues excludingprolines are modified.

In another embodiment of the invention, a method of expressing anantibody having CDRs designed by the aforementioned process is provided.In another embodiment, a pharmaceutical composition comprising anantibody that binds MCSF wherein said antibody is made using theaforementioned method is provided.

In still another embodiment of the invention, a method of preventing orreducing bone loss or remodeling is provided comprising administering toa subject afflicted with a disease that causes or contributes to orresults from osteolysis a therapeutically effective amount of any one ofthe aforementioned antibodies, thereby preventing or reducing bone lossor remodeling associated with the disease. In a related embodiment, amethod of treating a subject afflicted with a disease that causes orcontributes to or results from osteolysis is provided comprisingadministering to said subject a therapeutically effective amount of theantibody of any one of the aforementioned antibodies, thereby reducingthe severity of bone loss or remodeling associated with the disease.

In a related embodiment, the aforementioned method is provided whereinsaid disease is selected from the group consisting of metabolic bone orosteolytic diseases associated with relatively increased osteoclastactivity or the osteoclastic/osteoblastic process required for boneremodeling, including metabolic bone diseases, endocrinopathies(hypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, hyperthyroidism), hypercalcemia, deficiency states(rickets/osteomalacia, scurvy, malnutrition), chronic diseases(malabsorption syndromes, chronic renal failure (renal osteodystrophy),chronic liver disease (hepatic osteodystrophy)), drugs (glucocorticoids(glucocorticoid-induced osteoporosis), heparin, alcohol), and hereditarydiseases (osteogenesis imperfecta, homocystinuria), cancer,osteoporosis, -periprosthetic bone loss (for example, due to a foreignimplant in bone or joint replacement with a prosthesis, including totalhip arthroplasty), inflammation of bone associated with arthritis andrheumatoid arthritis, periodontal disease, fibrous dysplasia, and/orPaget's disease.

In still another embodiment, a method of preventing or treatingmetastatic cancer to bone is provided comprising administering to asubject afflicted with metastatic cancer a therapeutically effectiveamount of any one of the aforementioned antibodies. In a relatedembodiment, the method is provided wherein the metastatic cancer isbreast, lung, renal, multiple myeloma, thyroid, prostate,adenocarcinoma, blood cell malignancies, including leukemia andlymphoma; head and neck cancers; gastrointestinal cancers, includingesophageal cancer, stomach cancer, colon cancer, intestinal cancer,colorectal cancer, rectal cancer, pancreatic cancer, liver cancer,cancer of the bile duct or gall bladder; malignancies of the femalegenital tract, including ovarian carcinoma, uterine endometrial cancers,vaginal cancer, and cervical cancer; bladder cancer; brain cancer,including neuroblastoma; sarcoma, osteosarcoma; and skin cancer,including malignant melanoma or squamous cell cancer.

In yet another embodiment of the invention, a method of preventing boneloss or tumor growth is provided comprising administering to a subjectin need thereof therapeutically effective amounts of any one of theaforementioned antibodies.

Any one of the therapeutic methods described herein may includeadministering a second therapeutic agent. In still another relatedembodiment, the method is provided wherein the second therapeutic agentis a cancer chemotherapeutic agent or a bisphosphonate. In yet anotherrelated embodiment, the method is provided wherein the bisphonate iszeledronate, pamidronate, clodronate, etidronate, tilundronate,alendronate, or ibandronate. In yet another related embodiment, theaforementioned methods are provided wherein the therapeutic agent is acytotoxic chemotherapeutic agent. In another embodiment, theaforementioned method is provided wherein the subject is precluded fromreceiving bisphosphonate treatment.

In still another related embodiment, the aforementioned method isprovided wherein the antibody is effective to reduce the dosage ofsecond therapeutic agent required to achieve a therapeutic effect. Inanother embodiment, the second therapeutic agent is a non-M-CSF colonystimulating factor, for example G-CSF, or anti-RANKL antibody, orsoluble RANKL receptor.

In another embodiment of the invention, the aforementioned methods areprovided wherein the subject is a mammal. In a related embodiment, themammal is human.

In another embodiment, the aforementioned methods are provided whereinthe antibody inhibits the interaction between M-CSF and its receptor(M-CSFR). In another related embodiment, the antibody inhibitsosteoclast proliferation and/or differentiation induced by tumor cells.In yet another embodiment, the aforementioned methods are providedwherein the antibody is administered at a dose between about 1.0 μg/kgto about 100 mg/kg, 2 μg/kg to 30 mg/kg, 0.1 mg/kg to 30 mg/kg or 0.1mg/kg to 10 mg/kg body weight.

In another embodiment of the invention, the use of an antibody of theinvention is contemplated in the manufacture of a medicament forpreventing or reducing bone loss or remodeling in a patient exhibitingsymptoms of osteolysis or osteoclastic disease, and in the manufactureof a medicament for treating a patient afflicted with a disease thatcauses or contributes to or results from osteolysis or osteoclasticdisease. The aforementioned use is further contemplated wherein thedisease is selected from the group consisting of osteolytic diseasesassociated with relatively increased osteoclast activity or theosteoclastic/osteoblastic process required for bone remodeling,including metabolic bone diseases, endocrinopathies (includinghypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, hyperthyroidism), hypercalcemia, deficiency states(including rickets/osteomalacia, scurvy, malnutrition), chronic diseases(including malabsorption syndromes, chronic renal failure (includingrenal osteodystrophy), chronic liver disease (including hepaticosteodystrophy)), drugs (including glucocorticoids(glucocorticoid-induced osteoporosis), heparin, alcohol), and hereditarydiseases (including osteogenesis imperfecta, homocystinuria), cancer,osteoporosis, periprosthetic bone loss (for example, due to a foreignimplant in bone or joint replacement with a prosthesis, including totalhip arthroplasty), inflammation of bone associated with arthritis andrheumatoid arthritis, periodontal disease, fibrous dysplasia, and/orPaget's disease.

In another embodiment use of an antibody of the invention iscontemplated in the manufacture of a medicament for preventing ortreating metastatic cancer to bone in a patient suffering frommetastatic cancer. In a related embodiment, the metastatic cancer isbreast, lung, renal, multiple myeloma, thyroid, prostate,adenocarcinoma, blood cell malignancies, including leukemia or lymphoma;head or neck cancers; gastrointestinal cancers, including esophagealcancer, stomach cancer, colon cancer, intestinal cancer, colorectalcancer, rectal cancer, pancreatic cancer, liver cancer, cancer of thebile duct or gall bladder; malignancies of the female genital tract,including ovarian carcinoma, uterine endometrial cancers, vaginalcancer, or cervical cancer; bladder cancer; brain cancer, includingneuroblastoma; sarcoma, osteosarcoma; or skin cancer, includingmalignant melanoma or squamous cell cancer.

In still another embodiment, use of an antibody of the invention iscontemplated in the manufacture of a medicament for treating a patienthaving cancer.

In any of the aforementioned uses, the medicament is coordinated withtreatment using a second therapeutic agent. In a related embodiment, thesecond therapeutic agent is a cancer chemotherapeutic agent. In relatedembodiments, the second therapeutic agent is a non-M-CSF colonystimulating factor, or anti-RANKL antibody, or soluble RANKL receptor,or a bisphosphonate. In a related embodiment, the bisphonate iszeledronate, pamidronate, clodronate, etidronate, tilundronate,alendronate, or ibandronate.

In yet another embodiment of the invention, any of the aforementioneduses is contemplated wherein the patient is precluded from receivingbisphosphonate treatment, and/or wherein the patient has beenpre-treated with the second therapeutic agent. In a related embodiment,the second therapeutic agent is a cancer chemotherapeutic agent, anon-M-CSF colony stimulating factor, or anti-RANKL antibody, or solubleRANKL receptor, or a bisphosphonate. In yet another related embodiment,the bisphonate is zeledronate, pamidronate, clodronate, etidronate,tilundronate, alendronate, or ibandronate. In still another relatedembodiment, the patient is precluded from receiving bisphosphonatetreatment.

In another embodiment of the invention, the use of a synergisticcombination of an antibody of the invention for preparation of amedicament for treating a patient exhibiting symptoms of osteolysiswherein the medicament is coordinated with treatment using a secondtherapeutic agent is contemplated. In a related embodiment, the secondtherapeutic agent is a cancer chemotherapeutic agent, a non-M-CSF colonystimulating factor, or anti-RANKL antibody, or soluble RANKL receptor,or a bisphosphonate. In a related embodiment, the bisphonate iszeledronate, pamidronate, clodronate, etidronate, tilundronate,alendronate, or ibandronate. In still another embodiment, the patient isprecluded from receiving bisphosphonate treatment.

Embodiments of any of the aforementioned uses are contemplated whereinthe amount of antibody in the medicament is at a dose effective toreduce the dosage of second therapeutic agent required to achieve atherapeutic effect. In any of the aforementioned embodiments relating tobone loss or remodeling associated with cancer, the amount of antibodyin the medicament is preferably effective to inhibit osteoclastproliferation and/or differentiation induced by tumor cells.

The amount of antibody in any of the aforementioned medicaments may beat a dose between about 1.0 μg/kg to about 100 mg/kg or 2 μg/kg to 30mg/kg body weight. In a related embodiment, the amount of antibody inthe medicament is at a dose between about 0.1 mg/kg to 30 mg/kg bodyweight. In still another embodiment, the amount of antibody in themedicament is at a dose between about 0.1 mg/kg to 10 mg/kg body weight.

Kits are also contemplated by the present invention. In one embodiment,a kit comprising a therapeutically effective amount of an antibody ofthe invention, packaged in a container, such as a vial or bottle orprefilled syringe, and further comprising a label attached to orpackaged with the container, the label describing the contents of thecontainer and providing indications and/or instructions regarding use ofthe contents of the container to prevent or reduce bone loss isprovided. In another embodiment, a kit comprising a therapeuticallyeffective amount of an antibody of the invention, packaged in acontainer, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsThe container optionally further comprises a container with a secondtherapeutic agent and/or a container with suitable solution forreconstituting lyophilized antibody. The label may provide instructions,for example, regarding use of the contents of the container to a patientafflicted with a disease that causes or contributes to osteolysis or toprevent or reduce bone remodeling, to prevent or treat is provided. In arelated embodiment, the kit is provided wherein said disease is selectedfrom the group consisting of, or osteolytic diseases associated withrelatively increased osteoclast activity or theosteoclastic/osteoblastic process required for bone remodeling,including metabolic bone diseases, endocrinopathies (includinghypercortisolism, hypogonadism, primary or secondaryhyperparathyroidism, hyperthyroidism), hypercalcemia, deficiency states(including rickets/osteomalacia, scurvy, malnutrition), chronic diseases(including malabsorption syndromes, chronic renal failure (includingrenal osteodystrophy), chronic liver disease (including hepaticosteodystrophy)), drugs (including glucocorticoids(glucocorticoid-induced osteoporosis), heparin, alcohol), and hereditarydiseases (including osteogenesis imperfecta, homocystinuria), cancer,osteoporosis, periprosthetic bone loss (for example, due to a foreignimplant in bone or joint replacement with a prosthesis, including totalhip arthroplasty), inflammation of bone associated with arthritis andrheumatoid arthritis, periodontal disease, fibrous dysplasia, and/orPaget's disease. Such osteolytic disease associated with metastaticcancer to bone can result from breast, lung, renal, multiple myeloma,thyroid, prostate, adenocarcinoma, blood cell malignancies, includingleukemia or lymphoma; head or neck cancers; gastrointestinal cancers,including esophageal cancer, stomach cancer, colon cancer, intestinalcancer, colorectal cancer, rectal cancer, pancreatic cancer, livercancer, cancer of the bile duct or gall bladder; malignancies of thefemale genital tract, including ovarian carcinoma, uterine endometrialcancers, vaginal cancer, or cervical cancer; bladder cancer; braincancer, including neuroblastoma; sarcoma, osteosarcoma; or skin cancer,including malignant melanoma or squamous cell cancer.

In yet another embodiment, a kit is provided comprising atherapeutically effective amount of an antibody of the invention,packaged in a container, such as a vial or bottle or prefilled syringe,and further comprising a label attached to or packaged with thecontainer, the label describing the contents of the container andproviding indications and/or instructions regarding use of the contentsof the container to treat bone loss cancer.

In another embodiment, the kit further comprises a second therapeuticagent. In a related embodiment, the second therapeutic agent is a cancerchemotherapeutic agent, a non-M-CSF colony stimulating factor, oranti-RANKL antibody, or soluble RANKL receptor, or a bisphosphonate. Ina related embodiment, the bisphonate is zeledronate, pamidronate,clodronate, etidronate, tilundronate, alendronate, or ibandronate. Inyet another embodiment, the kit inludes instructions to treat a patientprecluded from receiving bisphosphonate treatment.

In another embodiment, the aforementioned kit is provided comprising adose of anti-M-CSF antibody effective to reduce the dosage of secondtherapeutic agent required to achieve a therapeutic effect. In anotherembodiment, the kit comprises a dose of anti-M-CSF antibody that issynergistic with the second therapeutic agent. In yet anotherembodiment, the kit comprises a dose of antibody effective to inhibitosteoclast proliferation and/or differentiation, induced by tumor cells.

In still another embodiment of the invention, the aforementioned kitcomprises a dose of antibody between about 1.0 μg/kg to about 100 mg/kgor 2 μg/kg to 30 mg/kg body weight. In another embodiment, the kitcomprises a dose of antibody between about 0.1 mg/kg to 30 mg/kg bodyweight. In still another embodiment, the kit comprises a dose ofantibody between about 0.1 mg/kg to 10 mg/kg body weight.

In still another embodiment of the invention, a method of preventingbone loss package, vial, prefilled syringe or container is providedcomprising a medicament comprising one or more of the aforementionedantibodies and instructions that the medicament should be used incombination with surgery or radiation therapy. In another embodiment, amethod of preventing or treating metastatic cancer to bone comprisingthe steps of administering any one of the aforementioned antibodies to asubject and treating the subject with surgery or radiation therapy isprovided. In another embodiment, a method of targeting a tumor cellexpressing membrane-bound M-CSF on its surface is provided comprisingthe step of administering any one of the aforementioned antibodies,wherein the antibody is conjugated to a radionuclide or other toxin. Inyet another embodiment, a method of treating a subject suffering from acancer is provided comprising administering a therapeutically effectiveamount of the any one of the aforementioned antibodies.

In still another embodiment of the invention, a method of preventingbone remodeling is provided comprising administering to a subjectafflicted with a disease that causes or contributes to osteolysis anamount of any one of the aforementioned antibodies in an amounteffective to neutralize M-CSF produced by the subject's cells, theamount being larger than the amount effective to neutralize M-CSFproduced by the cancer cells. In a related embodiment, a method oftreating a subject afflicted with a disease that causes or contributesto osteolysis is provided comprising administering to said subject anamount of any one of the aforementioned antibodies in an amounteffective to neutralize M-CSF produced by the subject's cells, theamount being larger than the amount effective to neutralize M-CSFproduced by the cancer cells.

In one embodiment of the invention, a pharmaceutical composition isprovided comprising antibody RX1, 5H4, MC1 and/or MC3, or a non-murineRX1, 5H4, MC1 and/or MC3 derived antibody or an RX1, 5H4, MC1 and/or MC3competing antibody, and a cancer therapeutic agent. In anotherembodiment of the invention, a package, vial or container is providedcomprising a medicament comprising antibody RX1, 5H4, MC1 and/or MC3, ora non-murine RX1, 5H4, MC1 and/or MC3 derived antibody or an RX1, 5H4,MC1 and/or MC3 competing antibody, and instructions that the medicamentshould be used in combination with surgery or radiation therapy.

In still another embodiment of the invention, a method of treating asubject suffering from a cancer is provided, wherein the cellscomprising the cancer do not secrete M-CSF, comprising the step ofadministering any one of the aforementioned antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a topology diagram showing the disulfide bonds in truncateddimeric M-CSF.

FIG. 2 is a stereodiagram of the C-alpha backbone of M-CSF with everytenth residue labeled and with the non-crystallographic symmetry axisindicated by a dotted line.

FIG. 3 is a comparison of osteoclast inducing activity between purifiedM-CSF and conditioned medium (CM) from MDA 231 cells and MCF7 cells.

FIG. 4A shows the amino acid sequence of M-CSF-specific murine antibodyRX1 (SEQ ID NOs: 2 and 4) (encoded by the cDNA insert of the plasmiddeposited with the American Type Culture Collection, Manassas, Va., USA,under ATCC deposit number PTA-6113) and a corresponding nucleic acidsequence (SEQ ID NOs: 1 and 3). The CDR regions are numbered and shownin bold.

FIGS. 4B and 4C show the amino acid sequences of M-CSF specific murineantibody RX1 light (SEQ ID NO: 5) and heavy chains (SEQ ID NO: 6),respectively, with high risk (bold), moderate risk (underline), and lowrisk residues identified according to Studnicka et al., WO93/11794.

FIG. 5A shows that M-CSF antibodies RX1 and 5A1 are species specific.FIG. 5B shows the M-CSF neutralization activity of antibodies MC1 andMC3.

FIG. 6 shows that antibody RX1 effectively inhibits osteolysis in ahuman xenograft model at a concentration 5 mg/kg.

FIG. 7 shows that the number of metastases is reduced when antibody RX1is administered to human breast cancer MDA-MB-231 bearing nude mice at aconcentration of 5 mg/kg.

FIGS. 8A and 8B shows that an M-CSF-specific antibody bound to breastcancer cell line MDA-MB-231 or to multiple myeloma cancer cell lineARH77.

FIG. 9 shows that M-CSF is prevalent on a number of cancer cellsurfaces.

FIG. 10 is the amino acid sequence of M-CSFα (SEQ ID NO: 7).

FIG. 11 is the amino acid sequence of M-CSFβ (SEQ ID NO: 8).

FIG. 12 is the amino acid sequence of M-CSFγ (SEQ ID NO: 9). A number ofpolymorphisms in the DNA sequence may result in amino acid differences.For example, a common polymorphism provides an Ala rather than Pro atposition 104.

FIGS. 13, 14, and 15 show the amino acid sequences of MCSF-specificmurine antibodies 5H4 (SEQ ID NOs: 10 and 11), MC1 (SEQ ID NOs: 12 and13) (produced by the hybridoma deposited under ATCC deposit numberPTA-6263) and MC3 (SEQ ID NOs: 14 and 15) (produced by the hybridomadeposited under ATCC deposit number PTA-6264), respectively.

FIGS. 16A and B are an alignment of CDR regions of the heavy and lightchain amino acid sequences of human M-CSF specific murine antibodiesRX1; 5H4; MC1; and MC3 (SEQ ID NOs: 16-38).

FIG. 17 shows the neutralization activities of intact versus Fabfragments for RX1 versus 5H4.

FIG. 18 shows the structure of M-CSF with RX1, 5H4, and MC3 epitopeshighlighted (SEQ ID NOs: 120, 122, and 123).

FIG. 19A shows (a) the risk line for the murine RX1 heavy chain (H=highrisk, M=moderate risk, L=low risk), (b) the RX1 heavy chain amino acidsequence (SEQ ID NO: 6), (c) the amino acid sequence of the closesthuman consensus sequence, Kabat Vh2 consensus, aligned to RX1 (SEQ IDNO: 39) and (d) changes that were made to produce two exemplary HumanEngineered™ sequences (SEQ ID NOs: 41 and 43). FIG. 19B shows the aminoacid sequences of the two exemplary heavy chain Human Engineered™sequences (SEQ ID NOs: 41 and 43), designated “low risk” and“low+moderate risk” as well as corresponding nucleic acid sequences (SEQID NOs: 40 and 42).

FIG. 20A shows (a) the risk line for the murine RX1 light chain (H=highrisk, M=moderate risk, L=low risk), (b) the RX1 light chain amino acidsequence (SEQ ID NO: 5), (c) the amino acid sequence of the closesthuman consensus sequence, Kabat Vk3 consensus, aligned to RX1 (SEQ EDNO: 49) and (d) changes that were made to produce two exemplary HumanEngineered™ sequences (SEQ ID NOs: 45 and 47). FIG. 20B shows the aminoacid sequences of the two exemplary light chain Human Engineered™sequences (SEQ ID NOs: 45 and 47), designated “low risk” and“low+moderate risk” as well as corresponding nucleic acid sequences (SEQID NOs: 44 and 46).

FIG. 21A shows (a) the risk line for the murine RX1 light chain (H=highrisk, M=moderate risk, L=low risk), (b) the RX1 light chain amino acidsequence (SEQ ID NO: 5), (c) the amino acid sequence of the closesthuman consensus sequence, Kabat Vk3 consensus, aligned to RX1 (SEQ IDNO: 49) and (d) an alternate exemplary amino acid sequence in whichpositions 54-56 were not changed (i.e. remained the murine sequence)(SEQ ID NO: 48). FIG. 21B shows the amino acid sequences of twoexemplary alternate light chain Human Engineered™ sequences (SEQ ID NOs:48, 136), as well as corresponding nucleic acid sequences (SEQ ID NOs:137 and 135).

FIG. 22A shows (a) the risk line for the murine RX1 light chain (H=highrisk, M=moderate risk, L=low risk), (b) the RX1 light chain amino acidsequence (SEQ ID NO: 5), (c) the amino acid sequence of the closesthuman consensus germline sequence, Vk6 Subgroup 2-1-(1) A14, aligned toRX1 (SEQ ID NO: 50) and (d) changes that were made to produce twoexemplary Human Engineered™ sequences (SEQ ID NOs: 51 and 53). FIG. 22Bshows the amino acid sequences of the two exemplary light chain HumanEngineered™ sequences (SEQ ID NOs: 51 and 53), designated “low risk” and“low+moderate risk” as well as the corresponding nucleic acid sequence(SEQ ID NO: 52).

FIGS. 23A and 23B show the alignment of murine RX1 light chain aminoacid sequence (SEQ ID NO: 54) with various human consensus and humangermline consensus sequences using the Kabat numbering system (aminoacid numbering indicated in line designated “POS”) (SEQ ID NOs: 55-82).

FIGS. 24A and 24B show the alignment of murine RX1 heavy chain aminoacid sequence (SEQ ID NO: 83) with various human consensus and humangermline consensus sequences using the Kabat numbering system (aminoacid numbering indicated in line designated “POS”) (SEQ ID NOs: 84-112).FIGS. 24C-24E show how the amino acid residues of antibodies 5H4, MC1and MC3 correspond to the Kabat numbering system (SEQ ID NOs: 10 and 11;SEQ ID NOs: 12 and 13; SEQ ID NOs: 14 and 15, respectively).

FIG. 25 shows the comparative neutralization of recombinant human M-CSFby recombinant murine RX1 antibody, labeled as rmRX1, and three versionsof Human Engineered™ RX1-1 antibody (in which all of the low riskchanges have been made) that each have a different constant region(IgG1, IgG2 or IgG4), labeled as heRX1-IG1, heRX1-1.G2, and heRX1-1.G4.

FIG. 26 shows the comparative neutralization of human serum byrecombinant murine RX1 antibody, labeled as rmRX1 and several differentversions of heRX1-1 (in which all of the low risk changes have beenmade) that each have a different constant region (IgG1, IgG2 or IgG4),labeled as RX2, RX1-1-IgG2, RX1-1-IgG1, RX1-1-IgG1, RX1-1-IgG4,RX1-a-IgG4.

FIG. 27 shows the comparative neutralization of MDA231 (breast cancercell line) medium by recombinant murine RX1 antibody, rmRX1, and severaldifferent versions of heRX1-1 (in which all of the low risk changes havebeen made) that each have a different constant region (IgG1, IgG2 orIgG4), labeled as RX2, RX1-1-IgG2, RX1-1-IgG1, RX1-1-IgG1, RX1-1-IgG4,RX1-a-IgG4.

FIG. 28 shows the effect on osteoclastogenesis (as measured by TRAPactivity) of recombinant murine RX1 antibody, rmRX1, and two differentversions heRX1-1 that each have a different constant region (Ig1 orIgG2), labeled heRX1-1.IgG1 and heRX1-1.IgG2.

FIG. 29A shows the amino acid (SEQ ID NO: 114) and nucleotide sequence(SEQ ID NO: 113) for heRX1-1.IgG1 with low risk amino acid changes. FIG.29B shows the amino acid (SEQ ID NO: 116) and nucleotide sequence (SEQID NO: 115) for heRX1-1.IgG1 with low+moderate risk amino' acid changes.

FIG. 30 shows the amino acid (SEQ ID NO: 119) and nucleotide sequence(cDNA (SEQ ID NO: 118) and genomic DNA (SEQ ID NO: 117)) forheRX1-1.IgG4 with low risk amino acid changes.

FIG. 31 shows the effect on osteoclastogenesis of 5H4.

DETAILED DESCRIPTION

The ability to metastasize is a defining characteristic of a cancer.Metastasis refers to the spread of cancer cells to other parts of thebody or to the condition produced by this spread. Metastasis is acomplex multi-step process that includes changes in the genetic materialof a cell, uncontrolled proliferation of the altered cell to form aprimary tumor, development of a new blood supply for the primary tumor,invasion of the circulatory system by cells from the primary tumor,dispersal of small clumps of primary tumor cells to other parts of thebody, and the growth of secondary tumors in those sites.

Bone is one of the most common sites of metastasis in human breast,lung, prostate and thyroid cancers, as well as other cancers, and inautopsies as many as 60% of cancer patients are found to have bonemetastasis. Osteolytic bone metastasis shows a unique step ofosteoclastic bone resorption that is not seen in metastasis to otherorgans. Bone loss associated with cancer metastasis is mediated byosteoclasts (multinucleated giant cells with the capacity to resorbmineralized tissues), which seem to be activated by tumor products.Activation of osteoclasts is associated with some cancers.

Colony stimulating factor (CSF-1), also known as macrophage colonystimulating factor (M-CSF), has been found crucial for osteoclastformation. In addition, M-CSF has been shown to modulate theosteoclastic functions of mature osteoclasts, their migration and theirsurvival in cooperation with other soluble factors and cell to cellinteractions provided by osteoblasts and fibroblasts (Fixe and Praloran,Cytokine 10: 3-7, 1998; Martin et al., Critical Rev. in Eukaryotic GeneExpression 8: 107-23 (1998)).

The full-length human M-CSF mRNA encodes a precursor protein of 554amino acids. Through alternative mRNA splicing and differentialpost-translational proteolytic processing, M-CSF can either be secretedinto the circulation as a glycoprotein or chondroitin sulfate containingproteoglycan or be expressed as a membrane spanning glycoprotein on thesurface of M-CSF producing cells. The three-dimensional structure of thebacterially expressed amino terminal 150 amino acids of human M-CSF, theminimal sequence required for full in vitro biological activity,indicates that this protein is a disulfide linked dimer with eachmonomer consisting of four alpha helical bundles and an anti-parallelbeta sheet (Pandit et al., Science 258: 1358-62 (1992)). Three distinctM-CSF species are produced through alternative mRNA splicing. The threepolypeptide precursors are M-CFSα of 256 amino acids, M-CSFβ of 554amino acids, and M-CSFγ of 438 amino acids. M-CSFβ is a secreted proteinthat does not occur in a membrane-bound form. M-CSFα is expressed as anintegral membrane protein that is slowly released by proteolyticcleavage. M-CSFα is cleaved at amino acids 191-197 of the sequence setout in FIG. 10. The membrane-bound form of M-CSF can interact withreceptors on nearby cells and therefore mediates specific cell-to-cellcontacts. The term “M-CSF” may also include amino acids 36-438 of FIG.12.

Various forms of M-CSF function by binding to its receptor M-CSFR (alsoknown as CSF-1R) on target cells. M-CSFR is a membrane spanning moleculewith five extracellular immunoglobulin-like domains, a transmembranedomain and an intracellular interrupted Src related tyrosine kinasedomain. M-CSFR is encoded by the c-fms proto-oncogene. Binding of M-CSFto the extracellular domain of M-CSFR leads to dimerization of thereceptor, which activates the cytoplasmic kinase domain, leading toautophosphorylation and phosphorylation of other cellular proteins(Hamilton J. A., J Leukoc Biol., 62(2):145-55 (1997); Hamilton J, A.,Immuno Today., 18(7): 313-7 (1997).

Phosphorylated cellular proteins induce a cascade of biochemical eventsleading to cellular responses: mitosis, secretion of cytokines, membraneruffling, and regulation of transcription of its own receptor (Fixe andPraloran, Cytokine 10: 32-37 (1998)).

M-CSF is expressed in stromal cells, osteoblasts, and other cells. It isalso expressed in breast, uterine, and ovarian tumor cells. The extentof expression in these tumors correlates with high grade and poorprognosis (Kacinski Ann. Med. 27: 79-85 (1995); Smith et al., Clin.Cancer Res. 1: 313-25 (1995)). In breast carcinomas, M-CSF expression isprevalent in invasive tumor cells as opposed to the intraductal(pre-invasive) cancer (Scholl et al., J. Natl. Cancer Inst. 86: 120-6(1994)). In addition, M-CSF is shown to promote progression of mammarytumors to malignancy (Lin et al., J. Exp. Med. 93: 727-39 (2001)). Forbreast and ovarian cancer, the production of M-CSF seems to beresponsible for the recruitment of macrophages to the tumor. Cancersarising in the prostate induce osteoblastic lesions in which new bone isformed as opposed to resorbed. In addition, prostate metastases areoften mixed, consisting of both osteolytic and osteoblastic lesions.Clinically, in some patients, there is an osteolytic phase that oftenprecedes the osteoblastic phase. The osteolytic phase often results inthe release of osteoblast growth factors which may help to “prime” theosteoblastic process. Recently, several studies have shown thatbisphosphonates, most commonly known to prevent/treat osteoporosis andother bone lytic diseases, can effectively prevent or inhibit growth ofosteoblastic metastases. In one embodiment of the present inventition,anti-M-CSF antibodies are used to treat either mixed-disease orosteoblastic disease. Thus, the present invention provides compositionsand methods for treating or preventing cancer, cancer metastasis andbone remodeling associated with cancer metastasis.

A preferred anti-M-CSF antibody murine RX1 was modified to be lessimmunogenic in humans based on the Human Engineering™ method ofStudnicka et al. In a preferred embodiment, 8 to 12 surface exposedamino acid residues of the heavy chain variable region and 16 to 19surface exposed residues in the light chain region were modified tohuman residues in positions determined to be unlikely to adverselyeffect either antigen binding or protein folding, while reducing itsimmunogenicity with respect to a human environment. Synthetic genescontaining modified heavy and/or light chain variable regions wereconstructed and linked to human r heavy chain and/or kappa light chainconstant regions. Any human heavy chain and light chain constant regionsmay be used in combination with the Human Engineered™ antibody variableregions. The human heavy and light chain genes were introduced intomammalian cells and the resultant recombinant immunoglobulin productswere obtained and characterized. Other exemplary anti-M-CSF antibodiessuch as 5H4, MC1, or MC3 are similarly Human Engineered™.

The term “RX 1-derived antibody” includes any one of the following:

1) an amino acid variant of murine antibody RX1 having the amino acidsequence set out in FIG. 4, including variants comprising a variableheavy chain amino acid sequence which is at least 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the aminoacid sequence as set forth in FIG. 4, and/or comprising a variable lightchain amino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90,91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the amino acidsequence as set forth in FIG. 4, taking into account similar amino acidsfor the homology determination;

2) M-CSF-binding polypeptides (excluding murine antibody RX 1)comprising one or more complementary determining regions (CDRs) ofmurine antibody RX 1 having the amino acid sequence set out in FIG. 4,preferably comprising at least CDR3 of the RX 1 heavy chain, andpreferably comprising two or more, or three or more, or four or more, orfive or more, or all six CDRs;

3) Human Engineered™ antibodies having the heavy and light chain aminoacid sequences set out in FIGS. 19B through 22B or variants thereofcomprising a heavy or light chain having at least 60% amino acidsequence identity with the original Human Engineered™ heavy or the lightchain of FIGS. 19B through 22B, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100% identical;

4) M-CSF-binding polypeptides (excluding murine antibody RX1) comprisingthe high risk residues of one or more CDRs of the Human Engineered™antibodies of FIGS. 19B through 22B, and preferably comprising high riskresidues of two or more, or three or more, or four or more, or five ormore, or all six CDRs; exemplary CDRs of Human Engineered™ RX1antibodies include SEQ ID NOs: 18, 21, 24, 32, 36 and QASQSIGTSIH (SEQID NO: 138);

5) Human Engineered™ antibodies or variants retaining the high riskamino acid residues set out in FIG. 4B, and comprising one or morechanges at the low or moderate risk residues set out in FIG. 4B;

-   -   for example, comprising one or more changes at a low risk        residue and conservative substitutions at a moderate risk        residue set out in FIG. 4B, or    -   for example, retaining the moderate and high risk amino acid        residues set out in FIG. 4B and comprising one or more changes        at a low risk residue,

where changes include insertions, deletions or substitutions and may beconservative substitutions or may cause the engineered antibody to becloser in sequence to a human light chain or heavy chain sequence, ahuman germline light chain or heavy chain sequence, a consensus humanlight chain or heavy chain sequence, or a consensus human germline lightchain or heavy chain sequence; that retain ability to bind M-CSF. Suchantibodies preferably bind to M-CSF with an affinity of at least 10⁻⁷,10⁻⁸ or 10⁻⁹ or higher and preferably neutralize the desired biologicalactivity of M-CSF, e.g. osteoclastogenesis-inducing activity of M-CSF.

Similarly, the term “MC3-derived antibody” includes any one of thefollowing:

1) an amino acid variant of murine antibody MC3 having the amino acidsequence set out in FIG. 15, including variants comprising a variableheavy chain amino acid sequence which is at least 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the aminoacid sequence as set forth in FIG. 15, and/or comprising a variablelight chain amino acid sequence which is at least 60, 65, 70, 75, 80,85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to the aminoacid sequence as set forth in FIG. 15, taking into account similar aminoacids for the homology determination;

2) M-CSF-binding polypeptides (optionally including or excluding murineantibody MC3) comprising one or more complementary determining regions(CDRs) of murine antibody MC3 having the amino acid sequence set out inFIG. 15, preferably comprising at least CDR3 of the MC3 heavy chain, andpreferably comprising two or more, or three or more, or four or more, orfive or more, or all six CDRs;

3) Human Engineered™ antibodies generated by altering the murinesequence according to the methods set forth in Studnicka et al., U.S.Pat. No. 5,766,886 and Example 5 herein, using the Rabat numbering setforth in FIGS. 24C-24E to identify low, moderate and high risk residues;such antibodies comprising at least one of the following heavy chainsand at least one of the following light chains: (a) a heavy chain inwhich all of the low risk residues have been modified, if necessary, tobe the same residues as a human reference immunoglobulin sequence or (b)a heavy chain in which all of the low and moderate risk residues havebeen modified, if necessary, to be the same residues as a humanreference immunoglobulin sequence, (c) a light chain in which all of thelow risk residues have been modified, if necessary, to be the sameresidues as a human reference immunoglobulin sequence or (b) a lightchain in which all of the low and moderate risk residues have beenmodified, if necessary, to be the same residues as a human referenceimmunoglobulin sequence;

4) variants of the aforementioned antibodies in preceding paragraph (3)comprising a heavy or light chain having at least 60% amino acidsequence identity with the original Human Engineered™ heavy or the lightchain, more preferably at least 80%, more preferably at least 85%, morepreferably at least 90%, and most preferably at least 95%, including forexample, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%identical;

5) M-CSF-binding polypeptides (optionally including or excluding murineantibody MC3) comprising the high risk residues of one or more CDRs ofthe murine MC3 antibody of FIG. 15, and preferably comprising high riskresidues of two or more, or three or more, or four or more, or five ormore, or all six CDRs;

6) Human Engineered™ antibodies or variants retaining the high riskamino acid residues of murine MC3 antibody, and comprising one or morechanges at the low or moderate risk residues;

-   -   for example, comprising one or more changes at a low risk        residue and conservative substitutions at a moderate risk        residue, or    -   for example, retaining the moderate and high risk amino acid        residues and comprising one or more changes at a low risk        residue,    -   where changes include insertions, deletions or substitutions and        may be conservative substitutions or may cause the engineered        antibody to be closer in sequence to a human light chain or        heavy chain sequence, a human germline light chain or heavy        chain sequence, a consensus human light chain or heavy chain        sequence, or a consensus human germline light chain or heavy        chain sequence; that retain ability to bind M-CSF. Such        antibodies preferably bind to M-CSF with an affinity of at least        10⁻⁷, 10⁻⁸ or 10⁻⁹ or higher and preferably neutralize the        desired biological activity of M-CSF, e.g.        osteoclastogenesis-inducing activity of M-CSF.

The term “5H4-derived antibody” or “MC1-derived antibody” is similarlydefined according to the above description.

As described in detail herein, anti-M-CSF antibodies such as RX1, 5H4,MC1 or MC3-derived antibodies, including Human Engineered™ antibodies orvariants, may be of different isotypes, such as IgG, IgA, IgM or IgE.Antibodies of the IgG class may include a different constant region,e.g. an IgG2 antibody may be modified to display an IgG1 or IgG4constant region. In preferred embodiments, the invention provides HumanEngineered™ antibodies or variants comprising a modified or unmodifiedIgG1 or IgG4 constant region. In the case of IgG1, modifications to theconstant region, particularly the hinge or CH2 region, may increase ordecrease effector function, including ADCC and/or CDC activity. In otherembodiments, an IgG2 constant region is modified to decreaseantibody-antigen aggregate formation. In the case of IgG4, modificationsto the constant region, particularly the hinge region, may reduce theformation of half-antibodies. In specific exemplary embodiments,mutating the IgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hingesequence Cys-Pro-Pro-Cys is provided.

Human Engineered™ antibodies containing IgG1 or IgG4 constant regionsare shown herein to have improved properties compared to HumanEngineered™ antibodies containing IgG2 constant regions. Choice of theIgG1 or IgG4 Fc region improved binding affinity, MCSF neutralizationactivity, and anti-osteoclast activity. In addition, choice of the IgG1or IgG4 Fc region provided antigen-antibody complexes that more closelyresembled those formed by the parent murine antibody.

The mobility at the hinge region thus appears to markedly affect bindingof antibody to the dimeric antigen MCSF as well as neutralizationactivity of the antibody. The invention contemplates generally thatpreparation of antibodies containing a heavy chain comprising a modifiedor unmodified IgG1 or IgG4 constant region, particularly the hinge andCH2 domains, or preferably at least the hinge domains, improves bindingaffinity and/or slows dissociation of antibody from dimeric antigens.

The term “RX1-competing antibody” includes

1) a non-murine or non-rodent monoclonal antibody that binds to the sameepitope of M-CSF as murine RX1 having the complete light and heavy chainsequences set out in FIG. 4;

2) a non-murine or non-rodent monoclonal antibody that binds to at least4 contiguous amino acids of amino acids 98-105 of the M-CSF of FIG. 12;and

3) a non-murine or non-rodent monoclonal antibody that competes withmurine antibody RX1 having the complete sequence set out in FIG. 4 forbinding to M-CSF, by more than 75%, more than 80%, or more than 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%.Such antibodies preferably bind to M-CSF with an affinity of at least10⁻⁷, 10⁻⁸ or 10⁻⁹ or higher and preferably neutralize the desiredbiological activity of M-CSF, e.g. osteoclastogenesis-inducing activityof M-CSF.

The term “MC1-competing antibody” or “MC3-competing antibody” or“5H4-competing antibody” is similarly defined with reference to themurine 5H4, MC1 or MC3 antibodies having the complete light and heavychain sequences set out in FIG. 13, 14 or 15, respectively, and withreference to the epitope of M-CSF bound by the antibody, e.g. aminoacids 65-73 or 138-144 of FIG. 12 (corresponding to M-CSF epitopesrecognized by 5H4 or MC3).

Optionally, any chimeric, human or humanized M-CSF antibody publiclydisclosed before the filing date hereof, or disclosed in an applicationfiled before the filing date hereof, is excluded from the scope of theinvention.

“Non-rodent” monoclonal antibody is any antibody, as broadly definedherein, that is not a complete intact rodent monoclonal antibodygenerated by a rodent hybridoma. Thus, non-rodent antibodiesspecifically include, but are not limited to, variants of rodentantibodies, rodent antibody fragments, linear antibodies, chimericantibodies, humanized antibodies, Human Engineered™ antibodies and humanantibodies, including human antibodies produced from transgenic animalsor via phage display technology. Similarly, non-murine antibodiesinclude but are not limited to variants of murine antibodies, murineantibody fragments, linear antibodies, chimeric, humanized, HumanEngineered™ and human antibodies.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include breast cancer, prostatecancer, colon cancer, squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, colorectal cancer, endometrial carcinoma, salivarygland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroidcancer, hepatic carcinoma and various types of head and neck cancer.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. Treatment ofpatients suffering from clinical, biochemical, radiological orsubjective symptoms of the disease, such as osteoblastic lesions orosteolysis, may include alleviating some or all of such symptoms orreducing the predisposition to the disease. The “pathology” of cancerincludes all phenomena that compromise the well being of the patient.This includes, without limitation, abnormal or uncontrollable cellgrowth, metastasis, interference with the normal functioning ofneighboring cells, release of cytokines or other secretory products atabnormal levels, suppression or aggravation of inflammatory orimmunological response, etc. Thus, improvement after treatment may bemanifested as decreased tumor size, decline in tumor growth rate,destruction of existing tumor cells or metastatic cells, and/or areduction in the size or number of metastases.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

As used herein, the phrase “metastatic cancer” is defined as cancersthat have potential to spread to other areas of the body, particularlybone. A variety of cancers can metastasize to the bone, but the mostcommon metastasizing cancers are breast, lung, renal, multiple myeloma,thyroid and prostate. By way of example, other cancers that have thepotential to metastasize to bone include but are not limited toadenocarcinoma, blood cell malignancies, including leukemia andlymphoma; head and neck cancers; gastrointestinal cancers, includingesophageal cancer, stomach cancer, colon cancer, intestinal cancer,colorectal cancer, rectal cancer, pancreatic cancer, liver cancer,cancer of the bile duct or gall bladder; malignancies of the femalegenital tract, including ovarian carcinoma, uterine endometrial cancers,vaginal cancer, and cervical cancer; bladder cancer; brain cancer,including neuroblastoma; sarcoma, osteosarcoma; and skin cancer,including malignant melanoma and squamous cell cancer. The presentinvention especially contemplates prevention and treatment oftumor-induced osteolytic lesions or mixed osteoblastic/osteolyticlesions in bone.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic M-CSF antibody thatwould be appropriate for an embodiment of the present invention, thatwill elicit the desired therapeutic or prophylactic effect or response,including alleviating some or all of such symptoms of disease orreducing the predisposition to the disease, when administered inaccordance with the desired treatment regimen.

Human “M-CSF” as used herein refers to a human polypeptide havingsubstantially the same amino acid sequence as the mature human M-CSFα,M-CSFβ, or M-CSFγ polypeptides described in Kawasaki et al., Science230:291 (1985), Cerretti et al., Molecular Immunology, 25:761 (1988), orLadner et al., EMBO Journal 6:2693 (1987), each of which areincorporated herein by reference. Such terminology reflects theunderstanding that the three mature M-CSFs have different amino acidsequences, as described above, and that the active form of M-CSF is adisulfide bonded dimer; thus, when the term “M-CSF” refers to thebiologically active form, the dimeric form is intended. “M-CSF dimer”refers to two M-CSF polypeptide monomers that have dimerized andincludes both homodimers (consisting of two of the same type of M-CSFmonomer) and heterodimers (consisting of two different monomers). M-CSFmonomers may be converted to M-CSF dimers in vitro as described in U.S.Pat. No. 4,929,700, which is incorporated herein by reference.

Anti-MCSF Antibodies

The present invention provides a M-CSF-specific antibody, such as RX1,5H4, MC1, and/or MC3, pharmaceutical formulations including aM-CSF-specific antibody, such as RX1, 5H4, MC1, and/or MC3, methods ofpreparing the pharmaceutical formulations, and methods of treatingpatients with the pharmaceutical formulations and compounds. The term“antibody” is used in the broadest sense and includes fully assembledantibodies, monoclonal antibodies, polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), antibody fragments that canbind antigen (e.g., Fab′, F′(ab)₂, Fv, single chain antibodies,diabodies), and recombinant peptides comprising the forgoing as long asthey exhibit the desired biological activity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that are typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe homogeneous culture, uncontaminated by other immunoglobulins withdifferent specificities and characteristics.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 [19751, or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624628[1991] end Marks et al., J. Mol.Biol., 222:581-597 (1991), for example.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes, IgA, IgD, IgE, IgG and IgM, and several ofthese may be further divided into subclasses or isotypes, e.g. IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains thatcorrespond to the different classes of immunoglobulins are called alpha,delta, epsilon, gamma and mu respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known. Different isotypes have different effector functions;for example, IgG1 and IgG3 isotypes have ADCC activity.

“Antibody fragments” comprise a portion of an intact full lengthantibody, preferably the antigen binding or variable region of theintact antibody. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies (Zapata et al.,Protein Eng., 8(10):1057-1062 (1995)); single-chain antibody molecules;and multispecific antibodies formed from antibody fragments. Papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize 35 readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two “Single-chain Fv” or “sFv” antibody fragments comprise the VHand VL domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains thatenables the Fv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a complementarity determiningregion or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of Immunological Interest, 5^(th) Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)]and/or those residues from a hypervariable loop (i.e., residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain as described by [Chothia et al., J. Mol. Biol. 196: 901-917(1987)].

“Framework” or FR residues are those variable domain residues other thanthe hypervariable region residues.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and 30 Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993).

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) monoclonal antibody including monoclonal, human, humanized,Human Engineered™ or variant anti-M-CSF antibodies having bindingspecificities for at least two different epitopes. Exemplary bispecificantibodies may bind to two different epitopes of M-CSF. Alternatively,an anti-M-CSF arm may be combined with an arm which binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI(CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the M-CSF-expressing cell. Bispecific antibodies may alsobe used to localize second therapeutic agents, e.g., cytotoxic agents tocells which express M-CSF. These antibodies possess an M-CSF-binding armand an arm which binds the cytotoxic agent (e.g., saporin,anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g., F(ab′).sub.2bispecific antibodies).

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. In yet afurther embodiment, Fab′-SH fragments directly recovered from E. colican be chemically coupled in vitro to form bispecific antibodies.(Shalaby et al., J. Exp. Med. 175:217-225 (1992)).

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992))The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments.

The fragments comprise a heavy chain variable region (V_(H)) connectedto a light-chain variable region (V_(L)) by a linker which is too shortto allow pairing between the two domains on the same chain. Accordingly,the V_(H) and V_(L) domains of one fragment are forced to pair with thecomplementary V_(L) and V_(H) domains of another fragment, therebyforming two antigen-binding sites. Another strategy for makingbispecific antibody fragments by the use of single-chain Fv (sFv) dimershas also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8(10):1057-1062(1995). Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60 (1991)).

In certain embodiments, the monoclonal, human, humanized, HumanEngineered™ or variant anti-M-CSF antibody is an antibody fragment, suchas an RX1, 5H4, MC1, or MC3 antibody fragment. Various techniques havebeen developed for the production of antibody fragments. Traditionally,these fragments were derived via proteolytic digestion of intactantibodies (see, e.g., Morimoto et al., Journal of Biochemical andBiophysical Methods 24:107-117 (1992) and Brennan et al., Science 229:81(1985)). However, these fragments can now be produced directly byrecombinant host cells. Better et al., Science 240: 1041-1043 (1988)disclose secretion of functional antibody fragments from bacteria (see,e.g., Better et al., Skerra et al. Science 240: 1038-1041 (1988)). Forexample, Fab′-SH fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). In another embodiment, the F(ab′)₂ isformed using the leucine zipper GCN4 to promote assembly of the F(ab′)₂molecule. According to another approach, Fv, Fab or F(ab′)₂ fragmentscan be isolated directly from recombinant host cell culture. Othertechniques for the production of antibody fragments will be apparent tothe skilled practitioner.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998),and U.S. Pat. No. 6,255,458, herein incorporated by reference in itsentirety. Briefly, the process for generating DNA encoding the heavy andlight chain immunoglobulin genes occurs primarily in developing B-cells.Prior to the rearranging and joining of various immunoglobulin genesegments, the V, D, J and constant (C) gene segments are found generallyin relatively close proximity on a single chromosome. DuringB-cell-differentiation, one of each of the appropriate family members ofthe V, D, J (or only V and J in the case of light chain genes) genesegments are recombined to form functionally rearranged heavy and lightimmunoglobulin genes. This gene segment rearrangement process appears tobe sequential. First, heavy chain D-to-J joints are made, followed byheavy chain V-to-DJ joints and light chain V-to-J joints.

The recombination of variable region gene segments to form functionalheavy and light chain variable regions is mediated by recombinationsignal sequences (RSS's) that flank recombinationally competent V, D andJ segments. RSS's necessary and sufficient to direct recombination,comprise a dyad-symmetric heptamer, an AT-rich nonamer and anintervening spacer region of either 12 or 23 base pairs. These signalsare conserved among the different loci and species that carry out D-J(or V-J) recombination and are functionally interchangeable. SeeOettinger, et al. (1990), Science, 248, 1517-1523 and references citedtherein. The heptamer comprises the sequence CACAGTG or its analoguefollowed by a spacer of unconserved sequence and then a nonamer havingthe sequence ACAAAAACC or its analogue. These sequences are found on theJ, or downstream side, of each V and D gene segment. Immediatelypreceding the germline D and J segments are again two recombinationsignal sequences, first the nonamer and then the heptamer againseparated by an unconserved sequence. The heptameric and nonamericsequences following a V_(L), V_(H) or D segment are complementary tothose preceding the J_(L), D or J_(H) segments with which theyrecombine. The spacers between the heptameric and nonameric sequencesare either 12 base pairs long or between 22 and 24 base pairs long.

In addition to the rearrangement of V, D and J segments, furtherdiversity is generated in the primary repertoire of immunoglobulin heavyand light chain by way of variable recombination at the locations wherethe V and J segments in the light chain are joined and where the D and Jsegments of the heavy chain are joined. Such variation in the lightchain typically occurs within the last codon of the V gene segment andthe first codon of the J segment. Similar imprecision in joining occurson the heavy chain chromosome between the D and J_(H) segments and mayextend over as many as 10 nucleotides. Furthermore, several nucleotidesmay be inserted between the D and J_(H) and between the V_(H) and D genesegments which are not encoded by genomic DNA. The addition of thesenucleotides is known as N-region diversity.

The net effect of such rearrangements in the variable region genesegments and the variable recombination which may occur during suchjoining is the production of a primary antibody repertoire.

“Fv” is the minimum antibody fragment that contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the VH VI dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)₂ antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem.

By “neutralizing antibody” is meant an antibody molecule that is able toeliminate or significantly reduce an effecter function of a targetantigen to which is binds. Accordingly, a “neutralizing” anti-targetantibody is capable of eliminating or significantly reducing an effecterfunction, such as enzyme activity, ligand binding, or intracellularsignaling.

As provided herein, the compositions for and methods of treatingosteolytic diseases (i.e. diseases associated with increasedosteoclastic activity), or cancer metastasis and/or bone remodelingassociated with cancer metastasis may utilize one or more antibody usedsingularly or in combination with other therapeutics to achieve thedesired effects. Antibodies according to the present invention may beisolated from an animal producing the antibody as a result of eitherdirect contact with an environmental antigen or immunization with theantigen. Alternatively, antibodies may be produced by recombinant DNAmethodology using one of the antibody expression systems well known inthe art (See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory (1988)). Such antibodies may includerecombinant IgGs, chimeric fusion proteins having immunoglobulin derivedsequences or “Human Engineered™” antibodies that may all be used for thetreatment of cancer metastasis and/or bone remodeling associated withcancer metastasis according to the present invention. In addition tointact, full-length molecules, the term “antibody” also refers tofragments thereof (such as, e.g., scFv, Fv, Fd, Fab, Fab′ and F(ab)′2fragments) or multimers or aggregates of intact molecules and/orfragments that bind to M-CSF (or M-CSFR). These antibody fragments bindantigen and may be derivatized to exhibit structural features thatfacilitate clearance and uptake, e.g., by incorporation of galactoseresidues.

In one embodiment of the present invention, M-CSF monoclonal antibodiesmay be prepared essentially as described in Halenbeck et al. U.S. Pat.No. 5,491,065 (1997), incorporated herein by reference. Exemplary M-CSFmonoclonal antibodies include those that bind to an apparentconformational epitope associated with recombinant or native dimericM-CSF with concomitant neutralization of biological activity. Theseantibodies are substantially unreactive with biologically inactive formsof M-CSF including monomeric and chemically derivatized dimeric M-CSF.

In other embodiments of the present invention, Human Engineered™anti-M-CSF monoclonal antibodies are provided. The phrase “HumanEngineered™ antibody” refers to an antibody derived from a non-humanantibody, typically a mouse monoclonal antibody. Alternatively, a HumanEngineered™ antibody may be derived from a chimeric antibody thatretains or substantially retains the antigen binding properties of theparental, non-human, antibody but which exhibits diminishedimmunogenicity as compared to the parental antibody when administered tohumans. The phrase “chimeric antibody,” as used herein, refers to anantibody containing sequence derived from two different antibodies (see,e.g., U.S. Pat. No. 4,816,567) which typically originate from differentspecies. Most typically, chimeric antibodies comprise human and murineantibody fragments, generally human constant and mouse variable regions.

The phrase “complementarity determining region” or the term “CDR” refersto amino acid sequences which together define the binding affinity andspecificity of the natural Fv region of a native immunoglobulin bindingsite (See, e.g., Chothia et al., J. Mol. Biol. 196:901 917 (1987); Kabatet al., U.S. Dept. of Health and Human Services NIH Publication No. 913242 (1991)). The phrase “constant region” refers to the portion of theantibody molecule that confers effector functions. In the presentinvention, mouse constant regions are preferably substituted by humanconstant regions. The constant regions of the subject antibodies arederived from human immunoglobulins. The heavy chain constant region canbe selected from any of the five isotypes: alpha, delta, epsilon, gammaor mu.

The antibodies of the present invention are said to be immunospecific orspecifically binding if they bind to antigen with a K_(a) of greaterthan or equal to about 10⁶M⁻¹ preferably greater than or equal to about10⁷M⁻¹, more preferably greater than or equal to about 10⁸M⁻¹, and mostpreferably greater than or equal to about 10⁹M⁻¹, 10¹⁰M⁻¹, or 10¹²M⁻¹.The anti-M-CSF antibodies may bind to different naturally occurringforms of M-CSF, including those expressed by the host's/subject'stissues as well as that expressed by the tumor. The monoclonalantibodies disclosed herein, such as RX1, 5H4, MC1, or MC3 antibody,have affinity for M-CSF and are characterized by a dissociationequilibrium constant (Kd) of at least 10⁻⁴ M, preferably at least about10⁻⁷M to about 10⁻⁸ M, more preferably at least about 10-⁸M, 10-¹⁰M,10-¹¹M or 10-¹²M. Such affinities may be readily determined usingconventional techniques, such as by equilibrium dialysis; by using theBIAcore 2000 instrument, using general procedures outlined by themanufacturer; by radioimmunoassay using ¹²⁵I labeled M-CSF; or byanother method known to the skilled artisan. The affinity data may beanalyzed, for example, by the method of Scatchard et al., Ann N.Y. Acad.Sci., 51:660 (1949). Thus, it will be apparent that preferred M-CSFantibodies will exhibit a high degree of specificity for M-CSF and willbind with substantially lower affinity to other molecules. Preferredantibodies bind M-CSF with a similar affinity as murine RX1 of FIG. 4binds to M-CSF, exhibit low immunogenicity, and exhibit the desiredbiological activity when tested in appropriate animal models of disease.Other exemplary antibodies bind M-CSF with a similar affinity as murine5H4, MC1 or MC3 of FIG. 13, 14 or 15, respectively, binds to M-CSF.

The antigen to be used for production of antibodies may be, e.g., intactM-CSF or a fragment of M-CSF that retains the desired epitope,optionally fused to another polypeptide that allows the epitope to bedisplayed in its native conformation. Alternatively, cells expressingM-CSF at their cell surface can be used to generate antibodies. Suchcells can be transformed to express M-CSF or may be other naturallyoccurring cells that express M-CSF. Other forms of M-CSF useful forgenerating antibodies will be apparent to those skilled in the art.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. At 7-14 days post-boosterinjection, the animals are bled and the serum is assayed for antibodytiter: Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same antigen, but conjugatedto a different protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001(1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Recombinant Production of Antibodies

DNA encoding the monoclonal antibodies may be isolated and sequencedfrom the hybridoma cells using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the monoclonal antibodies).Sequence determination will generally require isolation of at least aportion of the gene or cDNA of interest. Usually this requires cloningthe DNA or, preferably, mRNA (i.e., cDNA) encoding the monoclonalantibodies. Cloning is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library may be constructed by reverse transcriptionof polyA+ mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In a preferred embodiment, however, the polymerase chainreaction (PCR) is used to amplify cDNAs (or portions of full-lengthcDNAs) encoding an immunoglobulin gene segment of interest (e.g., alight chain variable segment). The amplified sequences can be readilycloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used not critical, so long as it ispossible to determine the sequence of some portion of the immunoglobulinpolypeptide of interest. As used herein, an “isolated” nucleic acidmolecule or “isolated” nucleic acid sequence is a nucleic acid moleculethat is either (1) identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the nucleic acid or (2) cloned, amplified,tagged, or otherwise distinguished from background nucleic acids suchthat the sequence of the nucleic acid of interest can be determined, isconsidered isolated. An isolated nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated nucleicacid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immunoglobulinpolypeptide, e.g., CDR regions, and the amplified sequences are insertedinto a phage vector. cDNAs encoding peptides of interest, e.g., variableregion peptides with desired binding characteristics, are identified bystandard techniques such as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library,or, when PCR is used, after subcloning the amplified sequence or bydirect PCR sequencing of the amplified segment. Sequencing is carriedout using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). By comparing thesequence of the cloned nucleic acid with published sequences of humanimmunoglobulin genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced, (i) the germline segmentusage of the hybridoma immunoglobulin polypeptide (including the isotypeof the heavy chain) and (ii) the sequence of the heavy and light chainvariable regions, including sequences resulting from N-region additionand the process of somatic mutation. One source of immunoglobulin genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, simian COScells, human embryonic kidney 293 cells (e.g., 293E cells), Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

Expression control sequences refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included. Where distinct designations are intended,it will be clear from the context.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

Amino acid sequence variants of the desired antibody may be prepared byintroducing appropriate nucleotide changes into the encoding DNA, or bypeptide synthesis. Such variants include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibodies. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe monoclonal, human, humanized, Human Engineered™ or variant antibody,such as changing the number or position of glycosylation sites.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

The invention also provides isolated nucleic acid encoding antibodies ofthe invention, optionally operably linked to control sequencesrecognized by a host cell, vectors and host cells comprising the nucleicacids, and recombinant techniques for the production of the antibodies,which may comprise culturing the host cell so that the nucleic acid isexpressed and, optionally, recovering the antibody from the host cellculture or culture medium.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

(1) Signal Sequence Component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. If prokaryotic host cells do not recognize and process thenative antibody signal sequence, the signal sequence may be substitutedby a signal sequence selected, for example, from the group of thepectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces a-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO90/13646. In mammalian cellexpression, mammalian signal sequences as welt as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(2) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter).

(3) Selective Marker Component

Expression and cloning vectors may contain a selective gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, tetracycline, G418, geneticin, histidinol, ormycophenolic acid (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs methotrexate, neomycin, histidinol, puromycin, mycophenolicacid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody-encoding nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody of the invention, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.Ura3-deficient yeast strains are complemented by plasmids bearing theura3 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al, Bio/Technology, 9: 968-975(1991).

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theantibody-encoding nucleic acid. Promoters suitable for use withprokaryotic hosts include the arabinose (e.g., araB) promoter phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody of the invention.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as Abelson leukemia virus, polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, most preferably, cytomegalovirus, a retrovirus, hepatitis-Bvirus, Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, from heat-shockpromoters, provided such promoters are compatible with the host cellsystems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibody-encodingsequence, but is preferably located at a site 5′ from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.Another is the mouse immunoglobulin light chain transcriptionterminator.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routineprocedure. Examples of useful mammalian host cell lines are Chinesehamster ovary cells, including CHOK1 cells (ATCC 15, CCL61), DXB-11,DG-44, and Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc.Natl. Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, [Graham etal., J. Gen Virol. 36: 59 (1977)]; baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies that target M-CSF.

(8) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S. Pat. Re. No.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as Gentamycin™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. Science 240: 1041-1043 (1988); ICSU ShortReports 10: 105 (1990); and Proc. Natl. Acad. Sci. USA 90: 457-461(1993) describe a procedure for isolating antibodies which are secretedto the periplasmic space of E. coli. (See also, [Carter et al.,Bio/Technology 10: 163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H) 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental mouse monoclonal antibodies, they can be used for thetreatment of humans with far less risk of anaphylaxis. Thus, theseantibodies may be preferred in therapeutic applications that involve invivo administration to a human.

Chimeric monoclonal antibodies, in which the variable Ig domains of amouse monoclonal antibody are fused to human constant Ig domains, can begenerated using standard procedures known in the art (See Morrison, S.L., et al. (1984) Chimeric Human Antibody Molecules; Mouse AntigenBinding Domains with Human Constant Region Domains, Proc. Natl. Acad.Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312,643-646. (1984)). Although some chimeric monoclonal antibodies haveproved less immunogenic in humans, the mouse variable Ig domains canstill lead to a significant human anti-mouse response.

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), or,alternatively, (2) transplanting the entire non-human variable domains,but “cloaking” them with a human-like surface by replacement of surfaceresidues (a process referred to in the art as “veneering”). In thepresent invention, humanized antibodies will include both “humanized”and “veneered” antibodies. These methods are disclosed in, e.g., Joneset al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad.Sci., U.S.A., 81:6851 6855 (1984); Morrison and Oi, Adv. Immunol., 44:6592 (1988); Verhoeyer et al., Science 239:1534 1536 (1988); Padlan,Molec. Immun. 28:489 498 (1991); Padlan, Molec. Immunol. 31(3):169 217(1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773 83 (1991)each of which is incorporated herein by reference.

In particular, a rodent antibody on repeated in vivo administration inman either alone or as a conjugate will bring about an immune responsein the recipient against the rodent antibody; the so-called HAMAresponse (Human Anti Mouse Antibody). The HAMA response may limit theeffectiveness of the pharmaceutical if repeated dosing is required. Theimmunogenicity of the antibody may be reduced by chemical modificationof the antibody with a hydrophilic polymer such as polyethylene glycolor by using the methods of genetic engineering to make the antibodybinding structure more human like. For example, the gene sequences forthe variable domains of the rodent antibody which bind CEA can besubstituted for the variable domains of a human myeloma protein, thusproducing a recombinant chimaeric antibody. These procedures aredetailed in EP 194276, EP 0120694, EP 0125023, EP 0171496, EP 0173494and WO 86/01533. Alternatively the gene sequences of the CDRs of therodent antibody may be isolated or synthesized and substituted for thecorresponding sequence regions of a homologous human antibody gene,producing a human antibody with the specificity of the original rodentantibody. These procedures are described in EP 023940, WO 90/07861 andWO91/09967. Alternatively a large number of the surface residues of thevariable domain of the rodent antibody may be changed to those residuesnormally found on a homologous human antibody, producing a rodentantibody which has a surface ‘veneer’ of residues and which willtherefore be recognized as self by the human body. This approach hasbeen demonstrated by Padlan et.al. (1991) Mol. Immunol. 28, 489.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriatefour framework regions of human variable Ig domains is also called CDRgrafting. This technique (Riechmann, L., et al., Nature 332, 323(1988)), utilizes the conserved framework regions (FR1-FR4) as ascaffold to support the CDR loops which are the primary contacts withantigen. A disadvantage of CDR grafting, however, is that it can resultin a humanized antibody that has a substantially lower binding affinitythan the original mouse antibody, because amino acids of the frameworkregions can contribute to antigen binding, and because amino acids ofthe CDR loops can influence the association of the two variable Igdomains. To maintain the affinity of the humanized monoclonal antibody,the CDR grafting technique can be improved by choosing human frameworkregions that most closely resemble the framework regions of the originalmouse antibody, and by site-directed mutagenesis of single amino acidswithin the framework or CDRs aided by computer modeling of the antigenbinding site (e.g., Co, M. S., et al. (1994), J. Immunol. 152,2968-2976).

One method of humanizing antibodies comprises aligning the non-humanheavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region which disturb the structure ofthe CDRs until the predicted conformation of the humanized sequencemodel closely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g., via Ashwellreceptors (See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 whichpatents are incorporated herein by reference).

A number of humanizations of mouse monoclonal antibodies by rationaldesign have been reported (See, for example, 20020091240 published Jul.11, 2002, WO 92/11018 and U.S. Pat. No., 5,693,762, U.S. Pat. No.5,766,866.

Amino Acid Sequence Variants

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis,” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody(including antibody fragment) fused to an epitope tag or a salvagereceptor epitope. Other insertional variants of the antibody moleculeinclude the fusion to a polypeptide which increases the serum half-lifeof the antibody, e.g. at the N-terminus or C-terminus.

The term “epitope tagged” refers to the antibody fused to an epitopetag. The epitope tag polypeptide has enough residues to provide anepitope against which an antibody there against can be made, yet isshort enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody there against does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Mol. Cell. Biol. 5(12): 3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody [Paborsky et al., Protein Engineering 3(6): 547-553 (1990)].Other exemplary tags are a poly-histidine sequence, generally around sixhistidine residues, that pelulits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions are shown inTable 1. The most conservative substitution is found under the headingof “preferred substitutions”. If such substitutions result in no changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Original Exemplary Preferred Residue Substitutions Ala (A) val;leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; glnarg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu(E) asp; gln asp Gly (G) ala His (H) asn; gln; lys; arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr Pro (P) ala Ser (S) thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve replacing an amino acid with anothermember of its class. Non-conservative substitutions involve replacing amember of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformationof the monoclonal, human, humanized, Human Engineered™ or variantantibody also may be substituted, generally with serine, to improve theoxidative stability of the molecule and prevent aberrant crosslinking.Conversely, cysteine bond(s) may be added to the antibody to improve itsstability (particularly where the antibody is an antibody fragment suchas an Fv fragment).

Affinity maturation involves preparing and screening antibody variantsthat have substitutions within the CDRs of a parent antibody andselecting variants that have improved biological properties such asbinding affinity relative to the parent antibody. A convenient way forgenerating such substitutional variants is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g. bindingaffinity).

Alanine scanning mutagenesis can be performed to identify hypervariableregion residues that contribute significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

Antibody variants can also be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example,deleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody. By way ofexample, the amino acids of RX1 at positions 41-43 of FIG. 4A (NGS) maybe retained. Alternatively, only amino acids 41 and 42 (NG) may beretained.

Ordinarily, amino acid sequence variants of the Human Engineered™antibody will have an amino acid sequence having at least 60% amino acidsequence identity with the original Human Engineered™ antibody aminoacid sequences of either the heavy or the light chain (e.g., as in anyof FIGS. 19B through 22B) more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, and most preferably at least95%, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the Human Engineered™ residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions (as defined in Table 1 above) as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. Thus, sequence identity canbe determined by standard methods that are commonly used to compare thesimilarity in position of the amino acids of two polypeptides. Using acomputer program such as BLAST or FASTA, two polypeptides are alignedfor optimal matching of their respective amino acids (either along thefull length of one or both sequences, or along a pre-determined portionof one or both sequences). The programs provide a default openingpenalty and a default gap penalty, and a scoring matrix such as PAM 250[a standard scoring matrix; see Dayhoff et al., in Atlas of ProteinSequence and Structure, vol. 5, supp. 3 (1978)] can be used inconjunction with the computer program. For example, the percent identitycan then be calculated as: the total number of identical matchesmultiplied by 100 and then divided by the sum of the length of thelonger sequence within the matched span and the number of gapsintroduced into the longer sequences in order to align the twosequences.

Other modifications of the antibody are contemplated. For example, itmay be desirable to modify the antibody of the invention with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodieswith enhanced biological activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., CancerResearch 53: 2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design 3: 219-230 (1989). In addition, it has beenshown that sequences within the CDR can cause an antibody to bind to MHCClass II and trigger an unwanted helper T-cell response. A conservativesubstitution can allow the antibody to retain binding activity yet loseits ability to trigger an unwanted T-cell response. Also see Steplewskiet al., Proc Natl Acad Sci USA. 1988; 85(13):4852-6, incorporated hereinby reference in its entirety, which described chimeric antibodieswherein a murine variable region was joined with human gamma 1, gamma 2,gamma 3, and gamma 4 constant regions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tissuetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half-life,for example, adding molecules such as PEG or other water solublepolymers, including polysaccharide polymers, to antibody fragments toincrease the half-life. This may also be achieved, for example, byincorporation of a salvage receptor binding epitope into the antibodyfragment (e.g., by mutation of the appropriate region in the antibodyfragment or by incorporating the epitope into a peptide tag that is thenfused to the antibody fragment at either end or in the middle, e.g., byDNA or peptide synthesis) (see, e.g., WO96/32478).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C.sub.L region orV.sub.L region, or both, of the antibody fragment. See alsoInternational applications WO 97/34631 and WO 96/32478 which describe Fcvariants and their interaction with the salvage receptor.

Thus, antibodies of the invention may comprise a human Fc portion, ahuman consensus Fc portion, or a variant thereof that retains theability to interact with the Fc salvage receptor, including variants inwhich cysteines involved in disulfide bonding are modified or removed,and/or in which the a met is added at the N-terminus and/or one or moreof the N-terminal 20 amino acids are removed, and/or regions thatinteract with complement, such as the Clq binding site, are removed,and/or the ADCC site is removed [see, e.g., Molec. Immunol. 29 (5):633-9 (1992)].

Previous studies mapped the binding site on human and murine IgG for FcRprimarily to the lower hinge region composed of IgG residues 233-239.Other studies proposed additional broad segments, e.g. Gly316-Lys338 forhuman Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lower hinge,e.g. Asn297 and Glu318 for murine IgG2b interacting with murine Fcreceptor II. The report of the 3.2-Å crystal structure of the human IgG1Fc fragment with human Fc receptor IIIA delineated IgG1 residuesLeu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor IIIA. It has been suggested based oncrystal structure that in addition to the lower hinge (Leu234-Gly237),residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues265-271) might play a role in binding to Fc receptor IIA. See Shields etal., J. Biol. Chem., 276(9):6591-6604 (2001), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptor bindingsites can result in altered effector function, such as altered ADCC orCDC activity, or altered half-life. As described above, potentialmutations include insertion, deletion or substitution of one or moreresidues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentIgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor 111A by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Va1303, Lys338, and Asp376. Variants thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA. See also Presta et al., Biochem. Soc. Trans.(2001) 30, 487-490.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes variants with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedClq binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcR11, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that Clq binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

United States Application Publication No. 20040132101, incorporated byreference herein in its entirety, describes variants with mutations atamino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325,328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240,241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297,298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering),of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325,327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding toan Fc gamma receptor.

Chappel et al., Proc Natl Acad Sci USA. 1991; 88(20):9036-40,incorporated herein by reference in its entirety, report that cytophilicactivity of IgG1 is an intrinsic property of its heavy chain CH2 domain.Single point mutations at any of amino acid residues 234-237 of IgG1significantly lowered or abolished its activity. Substitution of all ofIgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entire ELLGGPsequence (residues 233-238) was observed to be more active thanwild-type IgG1.

Isaacs et al., J. Immunol. 1998; 161(8):3862-9, incorporated herein byreference in its entirety, report that mutations within a motif criticalfor Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine234 to valine, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to alanineeliminated effector function of mouse IgG2b and also reduced the potencyof human IgG4.

Armour et al., Mol. Immunol. 2003; 40(9):585-93, incorporated byreference herein in its entirety, identified IgG1 variants which reactwith the activating receptor, FcgammaRIIa, at least 10-fold lessefficiently than wildtype IgG1 but whose binding to the inhibitoryreceptor, FcgammaRIIb, is only four-fold reduced. Mutations were made inthe region of amino acids 233-236 and/or at amino acid positions 327,330 and 331. See also WO 99/58572, incorporated by reference herein inits entirety.

Xu et al., J Biol Chem. 1994; 269(5):3469-74, incorporated by referenceherein in its entirety, report that mutating IgG1 Pro331 to Ser markedlydecreased Clq binding and visually eliminated lytic activity. Incontrast, the substitution of Pro for Ser331 in IgG4 bestowed partiallytic activity (40%) to the IgG4 Pro331 variant.

Schuurman et al., Mol Immunol. 2001; 38(1):1-8, incorporated byreference herein in its entirety, report that mutating one of the hingecysteines involved in the inter-heavy chain bond formation, Cys226, toserine resulted in a more stable inter-heavy chain linkage. Mutating theIgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent interactionbetween the heavy chains.

Angal et al., Mol Immunol. 1993; 30(1):105-8, incorporated by referenceherein in its entirety, report that mutating the serine at amino acidposition 241 in IgG4 to proline (found at that position in IgG1 andIgG2) led to the production of a homogeneous antibody, as well asextending serum half-life and improving tissue distribution compared tothe original chimeric IgG4.

Human and Human Engineered™ Antibodies

Human Engineering™

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position.

Variable regions of the light and heavy chains of a rodent antibody areHuman Engineered™ as follows to substitute human amino acids atpositions determined to be unlikely to adversely effect either antigenbinding or protein folding, but likely to reduce immunogenicity in ahuman environment. Amino acid residues that are at “low risk” positionsand that are candidates for modification according to the method areidentified by aligning the amino acid sequences of the rodent variableregions with a human variable region sequence. Any human variable regioncan be used, including an individual VH or VL sequence or a humanconsensus VH or VL sequence or an individual or consensus human germlinesequence. The amino acid residues at any number of the low riskpositions, or at all of the low risk positions, can be changed. Forexample, at each low risk position where the aligned murine and humanamino acid residues differ, an amino acid modification is introducedthat replaces the rodent residue with the human residue. Alternatively,the amino acid residues at all of the low risk positions and at anynumber of the moderate risk positions can be changed. Ideally, toachieve the least immunogenicity all of the low and moderate riskpositions are changed from rodent to human sequence.

Synthetic genes containing modified heavy and/or light chain variableregions are constructed and linked to human γ heavy chain and/or kappalight chain constant regions. Any human heavy chain and light chainconstant regions may be used in combination with the Human Engineered™antibody variable regions, including IgA (of any subclass, such as IgA1or IgA2), IgD, IgE, IgG (of any subclass, such as IgG1, IgG2, IgG3, orIgG4), or IgM. The human heavy and light chain genes are introduced intohost cells, such as mammalian cells, and the resultant recombinantimmunoglobulin products are obtained and characterized.

Human Antibodies from Transgenic Animals

Human antibodies to M-CSF can also be produced using transgenic animalsthat have no endogenous immunoglobulin production and are engineered tocontain human immunoglobulin loci. For example, WO 98/24893 disclosestransgenic animals having a human Ig locus wherein the animals do notproduce functional endogenous immunoglobulins due to the inactivation ofendogenous heavy and light chain loci. WO 91/741 also disclosestransgenic non-primate mammalian hosts capable of mounting an immuneresponse to an immunogen, wherein the antibodies have primate constantand/or variable regions, and wherein the endogenous immunoglobulinencoding loci are substituted or inactivated. WO 96/30498 discloses theuse of the Cre/Lox system to modify the immunoglobulin locus in amammal, such as to replace all or a portion of the constant or variableregion to form a modified antibody molecule. WO 94/02602 disclosesnon-human mammalian hosts having inactivated endogenous Ig loci andfunctional human Ig loci. U.S. Pat. No. 5,939,598 discloses methods ofmaking transgenic mice in which the mice lack endogenous heavy chains,and express an exogenous immunoglobulin locus comprising one or morexenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL 6, IL 8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667).

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immuno., 7:33 (1993); and U.S. Pat. No. 5,591,669, U.S. Pat. No.5,589,369, U.S. Pat. No. 5,545,807; and U.S Patent Application No.20020199213. U.S. Patent Application No. and 20030092125 describesmethods for biasing the immune response of an animal to the desiredepitope. Human antibodies may also be generated by in vitro activated Bcells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Human Antibodies from Phage Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. The antibodies produced by phagetechnology are produced as antigen binding fragments-usually

Fv or Fab fragments-in bacteria and thus lack effector functions.Effector functions can (be introduced by one of two strategies: Thefragments can be engineered either into complete antibodies forexpression in mammalian cells, or into bispecific antibody fragmentswith a second binding site capable of triggering an effector function.

Typically, the Fd fragment (V_(H)-C_(H)1) and light chain (V_(L)-C_(L))of antibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. By severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); and, Winter, G., et al., Annu.Rev. Immunol. 12, 433-455 (1994); U.S. patent application no.20020004215 and WO92/01047; U.S. patent application no. 20030190317published Oct. 9, 2003 and U.S. Pat. No. 6,054,287; U.S. Pat. No.5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193, and U.S. patent application no. 200120030044772published Mar. 6, 2003 describe methods for screening phage-expressedantibody libraries or other binding molecules by capture lift, a methodinvolving immobilization of the candidate binding molecules on a solidsupport.

The antibody products may be screened for activity and for suitabilityin the treatment methods of the invention using assays as described inthe section entitled “Screening Methods” herein or using any suitableassays known in the art.

Other Covalent Modifications

Covalent modifications of the antibody are also included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N.dbd.C.dbd.N—R′), where R and R′ aredifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the .alpha.-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulthydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Gene Therapy

Delivery of a therapeutic antibody to appropriate cells can be effectedvia gene therapy ex vivo, in situ, or in vivo by use of any suitableapproach known in the art, including by use of physical DNA transfermethods (e.g., liposomes or chemical treatments) or by use of viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus). Forexample, for in vivo therapy, a nucleic acid encoding the desiredantibody, either alone or in conjunction with a vector, liposome, orprecipitate may be injected directly into the subject, and in someembodiments, may be injected at the site where the expression of theantibody compound is desired. For ex vivo treatment, the subject's cellsare removed, the nucleic acid is introduced into these cells, and themodified cells are returned to the subject either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187. There area variety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, and calciumphosphate precipitation. A commonly used vector for ex vivo delivery ofa nucleic acid is a retrovirus.

Other in vivo nucleic acid transfer techniques include transfection withviral vectors (such as adenovirus, Herpes simplex I virus, oradeno-associated virus) and lipid-based systems. The nucleic acid andtransfection agent are optionally associated with a microparticle.Exemplary transfection agents include calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, commercialized as Lipofectin byGIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci. USA 84,7413-7417; Malone et al. (1989) Proc. Natl. Acad. Sci. USA 866077-6081); lipophilic glutamate diesters with pendent trimethylammoniumheads (Ito et al. (1990) Biochem. Biophys. Acta 1023, 124-132); themetabolizable parent lipids such as the cationic lipid dioctadecylamidoglycylspermine (DOGS, Transfectam, Promega) and dipalmitoylphosphatidylethanolamylspermine (DPPES) (J. P. Behr (1986) Tetrahedron Lett. 27,5861-5864; J. P. Behr et al. (1989) Proc. Natl. Acad. Sci. USA 86,6982-6986); metabolizable quaternary ammonium salts (DOTB,N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP)(Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters,ChoTB, ChoSC, DOSC)(Leventis et al. (1990) Biochim. Inter. 22, 235-241);3beta[N—(N′, N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol),dioleoylphosphatidyl ethanolamine(DOPE)/3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterolDC-Cholin one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065,8-14), spermine, spermidine, lipopolyamines (Behr et al., BioconjugateChem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al.,(1991) Biochim. Biophys. Acta 939, 8-18),[[(1,1,3,3-tetramethylbutyl)cre-soxy]ethoxy]ethyl]dimethylbenzylammoniumhydroxide (DEBDA hydroxide) with excess phosphatidylcholine/cholesterol(Ballas et al., (1988) Biochim. Biophys. Acta 939, 8-18),cetyltrimethylammonium bromide (CTAB)/DOPE mixtures (Pinnaduwage et al,(1989) Biochim. Biophys. Acta 985, 33-37), lipophilic diester ofglutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide(DDAB), and stearylamine in admixture with phosphatidylethanolamine(Rose et al., (1991) Biotechnique 10, 520-525), DDAB/DOPE (TransfectACE,GIBCO BRL), and oligogalactose bearing lipids. Exemplary transfectionenhancer agents that increase the efficiency of transfer include, forexample, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori NI et al, Biochem Biophys Res Commun Jun. 27, 1997; 235(3):726-9),chondroitan-based proteoglycans, sulfated proteoglycans,polyethylenimine, polylysine (Pollard H et al. J Biol Chem, 1998 273(13):7507-11), integrin-binding peptide CYGGRGDTP, linear dextrannonasaccharide, glycerol, cholesteryl groups tethered at the 3′-terminalinternucleoside link of an oligonucleotide (Letsinger, R. L. 1989 ProcNatl Acad Sci USA 86: (17):6553-6), lysophosphatide,lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

In some situations it may be desirable to deliver the nucleic acid withan agent that directs the nucleic acid-containing vector to targetcells. Such “targeting” molecules include antibodies specific for acell-surface membrane protein on the target cell, or a ligand for areceptor on the target cell. Where liposomes are employed, proteinswhich bind to a cell-surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake.Examples of such proteins include capsid proteins and fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. In other embodiments,receptor-mediated endocytosis can be used. Such methods are described,for example, in Wu et al., 1987 or Wagner et al., 1990. For review ofthe currently known gene marking and gene therapy protocols, seeAnderson 1992. See also WO 93/25673 and the references cited therein.For additional reviews of gene therapy technology, see Friedmann,Science, 244: 1275-1281 (1989); Anderson, Nature, supplement to vol.392, no 6679, pp. 25-30 (1998); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992).

Screening Methods

Effective therapeutics depend on identifying efficacious agents devoidof significant toxicity. Antibodies may be screened for binding affinityby methods known in the art. For example, gel-shift assays, Westernblots, radiolabeled competition assay, co-fractionation bychromatography, co-precipitation, cross linking, ELISA, and the like maybe used, which are described in, for example, Current Protocols inMolecular Biology (1999) John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety.

To initially screen for antibodies which bind to the desired epitope onM-CSF (e.g., those which block binding of RX1, 5H4, MC1 and/or MC3 toM-CSF), a routine cross-blocking assay such as that described inAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, EdHarlow and David Lane (1988), can be performed. Routine competitivebinding assays may also be used, in which the unknown antibody ischaracterized by its ability to inhibit binding of M-CSF to an M-CSFspecific antibody of the invention. Intact M-CSF, fragments thereof, orlinear epitopes such as represented by amino acids 98-105 of M-CSF ofFIG. 12, or amino acids 65-73 or 138-144 of FIG. 12 (corresponding toM-CSF epitopes recognized by 5H4 or MC3), can be used. Epitope mappingis described in Champe et al., J. Biol. Chem. 270: 1388-1394 (1995).

It is further contemplated that the antibodies are next tested for theireffect on osteoclastogenesis, followed by administration to animals.Compounds potentially useful in preventing or treating bone remodelingassociated with cancer metastasis may be screened using various assays.For instance, a candidate antagonist may first be characterized in acultured cell system to determine its ability to neutralize M-CSF ininducing osteoclastogenesis. Such a system may include the co-culture ofmouse calvarial osteoblasts and spleen cells (Suda et al., Modulation ofosteoclast differentiation. Endocr. Rev. 13: 66 80, 1992; Martin andUdagawa, Trends Endocrinol. Metab. 9: 6-12, 1998), the co-culture ofmouse stromal cell lines (e.g., MC3T3-G2/PA6 and ST2) and mouse spleencells (Udagawa et al., Endocrinology 125: 1805 13, 1989), and theco-culture of ST2 cells and bone marrow cells, peripheral bloodmononuclear cells or alveolar macrophages (Udagawa et al., Proc. Natl.Acad. Sci. USA 87: 7260 4, 1990; Sasaki et al., Cancer Res. 58: 462 7,1998; Mancino et al., J. Surg. Res.100: 18-24, 2001). In the absence ofany M-CSF antagonist, multinucleated cells formed in such co-culturessatisfy the major criteria of osteoclasts such as tartrate resistantacid phosphatase (TRAP, a marker enzyme of osteoclasts) activity,calcitonin receptors, p60C-STC, vitronectin receptors, and the abilityto form resorption pits on bone and dentine slices. The presence of aneffective M-CSF antagonist inhibits the formation of such multinucleatedcells.

In addition to the above co-culture systems, the ability of a candidateM-CSF antibody in inhibiting osteoclastogenesis may be assayed in astromal cell-free or osteoblast-free system. The M-CSF required forosteoclastogenesis may be provided by co-cultured metastatic cancercells (e.g., MDA 231) or conditioned medium from these cancer cells(Mancino et al., J. Surg. Res. 0: 18-24, 2001) or by addition ofpurified M-CSF.

Efficacy of a given M-CSF antibody in preventing or treating boneremodeling associated with cancer metastasis may also be tested in anyof the animal bone metastasis model systems familiar to those skilled inthe art. Such model systems include those involving direct injection oftumor cells into the medullary cavity of bones (Ingall, Proc. Soc. Exp.Biol. Med., 117: 819-22, 1964; Falasko, Clin. Orthop. 169: 20 7, 1982),into the rat abdominal aorta (Powles et al., Br. J. Cancer 28: 316 21,1973), into the mouse lateral tail vein or into the mouse left ventricle(Auguello et al., Cancer Res. 48: 6876 81, 1988). In the absence of aneffective M-CSF antagonist, osteolytic bone metastases fowled frominjected tumor cells may be determined by radiographs (areas ofosteolytic bone lesions) or histology and immunohistochemistry (bone andsoft tissues). Sasaki et al., Cancer Res. 55: 35517, 1995; Yoneda etal., J. Clin. Invest. 99: 2509 17, 1997. Clohisy and Ramnaraine, OrthopRes. 16: 660 6, 1998. Yin et al., J. Clin. Invest. 103: 197 206, 1999.In the presence of an effective M-CSF antibody, osteolytic bonemetastases may be prevented, or inhibited to result in fewer and/orsmaller metastases.

The M-CSF antibodies of the present invention may also be useful inpreventing or treating cancer metastasis. The effectiveness of acandidate M-CSF antibody in preventing or treating cancer metastasis Maybe screened using a human amnionic basement membrane invasion model asdescribed in Filderman et al., Cancer Res 52: 36616, 1992. In addition,any of the animal model systems for metastasis of various types ofcancers may also be used. Such model systems include, but are notlimited to, those described in Wenger et al., Clin. Exp. Metastasis 19:169 73, 2002; Yi et al., Cancer Res. 62: 917 23, 2002; Tsutsumi et al.,Cancer Lett 169: 77-85, 2001; Tsingotjidou et al., Anticancer Res. 21:971 8, 2001; Wakabayashi et al., Oncology 59: 75 80, 2000; Culp andKogerman, Front Biosci. 3:D672 83, 1998; Runge et al., Invest Radiol.32: 212 7; Shioda et al., J. Surg. Oncol. 64: 122 6, 1997; Ma et al.,Invest Opthalmol V is Sci. 37: 2293 301, 1996; Kuruppu et al., JGastroenterol Hepatol. 11: 26 32, 1996. In the presence of an effectiveM-CSF antibody, cancer metastases may be prevented, or inhibited toresult in fewer and/or smaller metastases.

The biological activity of a particular M-CSF antibody, or combinationof M-CSF antibodies, may be evaluated in vivo using a suitable animalmodel. For example, xenogenic lymphoma cancer models wherein humanlymphoma cells are introduced into immune compromised animals, such asnude or SCID mice. Efficacy may be predicted using assays which measureinhibition of tumor formation, tumor regression or metastasis, and thelike.

In one variation of an in vitro assay, the invention provides a methodcomprising the steps of (a) contacting an immobilized M-CSF with acandidate antibody and (b) detecting binding of the candidate antibodyto the M-CSF. In an alternative embodiment, the candidate antibody isimmobilized and binding of M-CSF is detected. Immobilization isaccomplished using any of the methods well known in the art, includingcovalent bonding to a support, a bead, or a chromatographic resin, aswell as non-covalent, high affinity interaction such as antibodybinding, or use of streptavidin/biotin binding wherein the immobilizedcompound includes a biotin moiety. Detection of binding can beaccomplished (i) using a radioactive label on the compound that is notimmobilized, (ii) using a fluorescent label on the non-immobilizedcompound, (iii) using an antibody immunospecific for the non-immobilizedcompound, (iv) using a label on the non-immobilized compound thatexcites a fluorescent support to which the immobilized compound isattached, as well as other techniques well known and routinely practicedin the art.

Antibodies that modulate (i.e., increase, decrease, or block) theactivity or expression of M-CSF may be identified by incubating aputative modulator with a cell expressing a M-CSF and determining theeffect of the putative modulator on the activity or expression of theM-CSF. The selectivity of an antibody that modulates the activity of aM-CSF polypeptide or polynucleotide can be evaluated by comparing itseffects on the M-CSF polypeptide or polynucleotide to its effect onother related compounds. Selective modulators may include, for example,antibodies and other proteins, peptides, or organic molecules whichspecifically bind to M-CSF polypeptides or to a nucleic acid encoding aM-CSF polypeptide. Modulators of M-CSF activity will be therapeuticallyuseful in treatment of diseases and physiological conditions in whichnormal or aberrant activity of M-CSF polypeptide is involved.

The invention also comprehends high throughput screening (HTS) assays toidentify antibodies that interact with or inhibit biological activity(i.e., inhibit enzymatic activity, binding activity, etc.) of a M-CSFpolypeptide. HTS assays permit screening of large numbers of compoundsin an efficient manner. Cell-based HTS systems are contemplated toinvestigate the interaction between M-CSF polypeptides and their bindingpartners. HTS assays are designed to identify “hits” or “lead compounds”having the desired property, from which modifications can be designed toimprove the desired property. Chemical modification of the “hit” or“lead compound” is often based on an identifiable structure/activityrelationship between the “hit” and M-CSF polypeptides.

Another aspect of the present invention is directed to methods ofidentifying antibodies which modulate (i.e., decrease) activity of aM-CSF comprising contacting a M-CSF with an antibody, and determiningwhether the antibody modifies activity of the M-CSF. The activity in thepresence of the test antibody is compared to the activity in the absenceof the test antibody. Where the activity of the sample containing thetest antibody is lower than the activity in the sample lacking the testantibody, the antibody will have inhibited activity.

A variety of heterologous systems is available for functional expressionof recombinant polypeptides that are well known to those skilled in theart. Such systems include bacteria (Strosberg, et al., Trends inPharmacological Sciences (1992) 13:95-98), yeast (Pausch, Trends inBiotechnology (1997) 15:487-494), several kinds of insect cells (VandenBroeck, Int. Rev. Cytology (1996) 164:189-268), amphibian cells(Jayawickreme et al., Current Opinion in Biotechnology (1997) 8:629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; seeGerhardt, et al., Eur. J. Pharmacology (1997) 334:1-23). These examplesdo not preclude the use of other possible cell expression systems,including cell lines obtained from nematodes (PCT application WO98/37177).

In one embodiment of the invention, methods of screening for antibodieswhich modulate the activity of M-CSF comprise contacting test antibodieswith a M-CSF polypeptide and assaying for the presence of a complexbetween the antibody and the M-CSF. In such assays, the ligand istypically labeled. After suitable incubation, free ligand is separatedfrom that present in bound form, and the amount of free or uncomplexedlabel is a measure of the ability of the particular antibody to bind tothe M-CSF or M-CSFR polypeptide

In another embodiment of the invention, high throughput screening forantibody fragments or CDRs having suitable binding affinity to a M-CSFpolypeptide is employed. Briefly, large numbers of different smallpeptide test compounds are synthesized on a solid substrate. The peptidetest antibodies are contacted with a M-CSF polypeptide and washed. BoundM-CSF polypeptides are then detected by methods well known in the art.Purified polypeptides of the invention can also be coated directly ontoplates for use in the aforementioned drug screening techniques. Inaddition, non-neutralizing antibodies can be used to capture the proteinand immobilize it on the solid support.

Combination Therapy

Having identified more than one M-CSF antibody that is effective in ananimal model, it may be further advantageous to mix two or more suchM-CSF antibodies together to provide still improved efficacy againstbone loss or against cancer metastasis and/or bone remodeling associatedwith cancer metastasis. Compositions comprising one or more M-CSFantibody may be administered to persons or mammals suffering from, orpredisposed to suffer from, bone loss or cancer metastasis and/or boneremodeling associated with cancer metastasis. Concurrent administrationof two therapeutic agents does not require that the agents beadministered at the same time or by the same route, as long as there isan overlap in the time period during which the agents are exerting theirtherapeutic effect. Simultaneous or sequential administration iscontemplated, as is administration on different days or weeks.

Although M-CSF antibody therapy may be useful for all stages of cancers,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Combining the antibody therapy method achemotherapeutic or radiation regimen may be preferred in patients thathave not received chemotherapeutic treatment, whereas treatment with theantibody therapy may be indicated for patients who have received one ormore chemotherapies. Additionally, antibody therapy can also enable theuse of reduced dosages of concomitant chemotherapy, particularly inpatients that do not tolerate the toxicity of the chemotherapeutic agentvery well.

The method of the invention contemplate the administration of singleanti-M-CSF antibodies, as well as combinations, or “cocktails”, ofdifferent antibodies. Such antibody cocktails may have certainadvantages inasmuch as they contain antibodies which exploit differenteffector mechanisms or combine directly cytotoxic antibodies withantibodies that rely on immune effector functionality. Such antibodiesin combination may exhibit synergistic therapeutic effects.

Combining RX1 or Human Engineered™ derivative of RX1 antibody with othertherapeutics can have an effect on a patient experiencing osteoclasticdisease and/or tumor growth or metastasis. For example, one could use ofRX1 antibody in the manufacture of a medicament for treating a patienthaving an osteolytic disease wherein said medicament is coordinated withtreatment using an anti-RANKL antibody, soluble RANKL receptor, otherRANKL inhibitors, or bisphosphonates (e.g., Aredia; Zometa; Clodronate).Alternatively, one could use an anti-RANKL antibody or bisphosphonate inthe manufacture of a medicament for treating a for treating a patienthaving an osteolytic disease wherein said medicament coordinated withtreatment using RX1 antibody or human engineered derivative of RX1antibody. The combination might also have a synergistic effect in atreated patient. The RX1 antibody and other therapeutic need not beadministered simultaneously. RX1 or the human engineered variant and theother therapeutic can administered within 1 day, 1 week, 2 weeks, 4weeks, 2 months, 3 months, 6 months, 1 year or two years of each other.

The invention also contemplates the use of a RX1 antibody or a HumanEngineered™ derivative of RX1 antibody in the manufacture of amedicament for treating a patient having an osteolytic disease whereinsaid medicament is used in a patient that has been pre-treated with ananti-RANKL antibody or bisphosphonates. “Pre-treatment” means that apatient had been treated within 2 years, 1 year, 6 months, 3 months 2months, 1 month, 2 weeks, 1 week, or at least one day one beforetreatment with RX1 or Human Engineered™ variant of RX1.

RX1 antibody or Human Engineered™ variants can be used in combinationwith other cancer therapeutics. For example, one could use RX1 antibodyor human engineered variants in the manufacture of a medicament fortreating a patient having cancer disease wherein said medicament iscoordinated with treatment using other therapeutic agents and/orprocedures, including but not limited to various chemotherapeuticagents, androgen-blockers, and immune modulators (e.g., IL-2, GM-CSF,SLC), Bisphosphonate(s) (e.g., Aredia; Zometa; Clodronate), surgery,radiation, cytotoxic chemotherapy, hormone therapy (e.g., Tamoxifen;anti-Androgen therapy), antibody therapy (e.g., antibodies to RANKL/RANKneutralizing; PTHrP neutralizing, anti-Her2, anti-CD20, anti-CD40, CD22,VEGF, IGFR-1, EphA2, HAAH, TMEFF2, CAIX antibodies), therapeutic proteintherapy (e.g., soluble RANKL receptor; OPG, and PDGF and MMPinhibitors), small molecule drug therapy (e.g., Src-kinase inhibitor),kinase inhibitors of growth factor receptors, or RANKL inhibitors,oligonucleotides therapy (e.g., RANKL or RANK or PTHrP Anti-sense), genetherapy (e.g., RANKL or RANK inhibitors), peptide therapy (e.g. muteinsof RANKL) as well as those proteins, peptides, compounds, and smallmolecules described herein. Treatment of mixed osteolytic/osteoblasticlesions may include use of RX1 antibody and another therapeutic agentthat inhibits osteoblastogenesis, including, for example, a bonemorphogenetic protein (BMP) antagonist, such as a BMP-2 antagonist, atransforming growth factor-β1 (TGFβ1) antagonist, a transfoiming growthfactor-β2 TGFβ2 antagonist, an insulin-like growth factor 1 (IGF1)antagonist, an insulin-like growth factor 2 (IGF2) antagonist, afibroblast growth factor (FGF) antagonist, a platelet-derived growthfactor (PDGF) antagonist, a WNT antagonist such as DICKKOPF-1 (DKK-1), aβ-catenin antagonist, an osterix antagonist, a core binding factor 1(RUNX2)antagonist, and an endothelin receptor antagonist such asatrasentan.

RX1 and Human Engineered™ variants can be used in the manufacture of amedicament for treating patients that have been pretreated with theabove mentioned therapeutics.

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g.,I¹³¹, I¹²⁵, Y⁹⁰ andRe¹⁸⁶), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the invention.

Cancer chemotherapeutic agents include, without limitation, alkylatingagents, such as carboplatin and cisplatin; nitrogen mustard alkylatingagents; nitrosourea alkylating agents, such as carmustine (BCNU);antimetabolites, such as methotrexate; folinic acid; purine analogantimetabolites, mercaptopurine; pyrimidine analog antimetabolites, suchas fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as aldesleukin, interleukin-2, docetaxel,etoposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin(ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycinsincluding mitomycin C; and vinca alkaloid natural antineoplastics, suchas vinblastine, vincristine, vindesine; hydroxyurea; aceglatone,adriamycin, Ifosfamide, enocitabine, epitiostanol, aclarubicin,ancitabine, nimustine, procarbazine hydrochloride, carboquone,carboplatin, carmofur, chromomycin A3, antitumor polysaccharides,antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan,cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa,tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin,caminomycin, esperamicins (See, e.g., U.S. Pat. No. 4,675,187),neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan,honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane,mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolorextract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

Further, additional agents used as therapy for cancer patients includeEPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine(AZT); interleukins 1 through 18, including mutants and analogues;interferons or cytokines, such as interferons α,β, and γ hormones, suchas luteinizing hormone releasing hormone (LHRH) and analogues and,gonadotropin releasing hormone (GnRH); growth factors, such astransforming growth factor-β (TGF-β), fibroblast growth factor (FGF),nerve growth factor (NGF), growth hormone releasing factor (GHRF),epidermal growth factor (EGF), fibroblast growth factor homologousfactor (FGFHF), hepatocyte growth factor (HGF), and insulin growthfactor (IGF); tumor necrosis factor-α& β (TNF-α& β); invasion inhibitingfactor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD);complement factors; anti-angiogenesis factors; antigenic materials; andpro-drugs.

Prodrug refers to a precursor or derivative form of a pharmaceuticallyactive substance that is less cytotoxic or non-cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into an active or the more active parent form.See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for useherein include, but are not limited to, those chemotherapeutic agentsdescribed above.

Administration and Preparation

The anti-M-CSF antibodies used in the practice of a method of theinvention may be formulated into pharmaceutical compositions comprisinga carrier suitable for the desired delivery method. Suitable carriersinclude any material which, when combined with the anti-M-CSFantibodies, retains the desired activity of the antibody and isnonreactive with the subject's immune systems. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine and the like, and may includeother proteins for enhanced stability, such as albumin, lipoprotein,globulin, etc., subjected to mild chemical modifications or the like.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEENT™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the antibody is suitably administered bypulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections. Other administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site.

For nasal administration, the pharmaceutical formulations andmedicaments may be a spray or aerosol containing an appropriatesolvent(s) and optionally other compounds such as, but not limited to,stabilizers, antimicrobial agents, antioxidants, pH modifiers,surfactants, bioavailability modifiers and combinations of these. Apropellant for an aerosol formulation may include compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils may be employed as solvents or suspendingagents. Preferably, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intennolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions. Other strategiesknown in the art may be used.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carries are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, genotype, sex, and diet ofthe subject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

M-CSF antibodies useful as therapeutics for osteolytic diseases,including cancer metastasis to bone and bone remodeling associated withsuch diseases, will often be prepared substantially free of othernaturally occurring immunoglobulins or other biological molecules.Preferred M-CSF antibodies will also exhibit minimal toxicity whenadministered to a mammal afflicted with, or predisposed to suffer fromosteolytic diseases.

The compositions of the invention may be sterilized by conventional,well known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride andstabilizers (e.g., 1 20% maltose, etc.).

The M-CSF antibodies of the present invention may also be administeredvia liposomes, which are small vesicles composed of various types oflipids and/or phospholipids and/or surfactant which are useful fordelivery of a drug (such as the antibodies disclosed herein and,optionally, a chemotherapeutic agent). Liposomes include emulsions,foams, micelles, insoluble monolayers, phospholipid dispersions,lamellar layers and the like, and can serve as vehicles to target theM-CSF antibodies to a particular tissue as well as to increase the halflife of the composition. A variety of methods are available forpreparing liposomes, as described in, e.g., U.S. Pat. Nos. 4,837,028 and5,019,369, which patents are incorporated herein by reference.

Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980);and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556. Particularlyuseful liposomes can be generated by the reverse phase evaporationmethod with a lipid composition comprising phosphatidylcholine,cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).Liposomes are extruded through filters of defined pore size to yieldliposomes with the desired diameter. Fab′ fragments of the antibody ofthe present invention can be conjugated to the liposomes as described inMartin et al., J. Biol. Chem. 257: 286-288 (1982) via a disulfideinterchange reaction. A chemotherapeutic agent (such as Doxorubicin) isoptionally contained within the liposome [see, e.g., Gabizon et al., J.National Cancer Inst. 81(19): 1484 (1989)].

The concentration of the M-CSF antibody in these compositions can varywidely, i.e., from less than about 10%, usually at least about 25% to asmuch as 75% or 90% by weight and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected. Actual methods for preparing orally, topicallyand parenterally administrable compositions will be known or apparent tothose skilled in the art and are described in detail in, for example,Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co.,Easton, Pa. (1995), which is incorporated herein by reference.

Determination of an effective amount of a composition of the inventionto treat disease in a patient can be accomplished through standardempirical methods which are well known in the art. For example, the invivo neutralizing activity of sera from a subject treated with a givendosage of M-CSF antibody may be evaluated using an assay that determinesthe ability of the sera to block M-CSF induced proliferation andsurvival of murine monocytes (CD11b+ cell, a subset of CD11 cells, whichexpresses high levels of receptor to M-CSF) in vitro as described inCenci et al., J Clin. Invest. 1055: 1279-87, 2000.

Compositions of the invention are administered to a mammal alreadysuffering from, or predisposed to or at risk of an osteolytic disease,including cancer metastasis to bone and bone remodeling associated withsuch diseases, in an amount sufficient to prevent or at least partiallyarrest the development of disease. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Effective amounts of aM-CSF antibody will vary and depend on the severity of the disease andthe weight and general state of the patient being treated, but generallyrange from about 1.0 μg/kg to about 100 mg/kg body weight. Exemplarydoses may range from about 10 μg/kg to about 30 mg/kg, or from about 0.1mg/kg to about 20 mg/kg or from about 1 mg/kg to about 10 mg/kg perapplication. Antibody may also be dosed by body surface area (e.g. up to4.5 g/square meter). Other exemplary doses of antibody include up to 8 gtotal in a single administration (assuming a body weight of 80 kg orbody surface area of 1.8 square meters).

Administration may be by any means known in the art. For example,antibody may be administered by one or more separate bolusadministrations, or by short or long term infusion over a period of,e.g., 5, 10, 15, 30, 60, 90, 120 minutes or more. Following an initialtreatment period, and depending on the patient's response and toleranceof the therapy, maintenance doses may be administered, e.g., weekly,biweekly, every 3 weeks, every 4 weeks, monthly, bimonthly, every 3months, or every 6 months, as needed to maintain patient response. Morefrequent dosages may be needed until a desired suppression of diseasesymptoms occurs, and dosages may be adjusted as necessary. The progressof this therapy is easily monitored by conventional techniques andassays. The therapy may be for a defined period or may be chronic andcontinue over a period of years until disease progression or death.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage of antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

In any event, the formulations should provide a quantity of M-CSFantibody over time that is sufficient to effectively prevent or minimizethe severity of osteolytic disease, including cancer metastasis to boneand bone remodeling associated with such diseases. The compositions ofthe present invention may be administered alone or as an adjunct therapyin conjunction with other therapeutics known in the art for thetreatment of such diseases.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thetherapeutically effective amount of the antibody to be administered willbe governed by such considerations, and is the minimum amount necessaryto prevent, ameliorate, or treat the M-CSF mediated disease, conditionor disorder. Such amount is preferably below the amount that is toxic tothe host or renders the host significantly more susceptible toinfections.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disease, condition or disorderor treatment, and other factors discussed above. These are generallyused in the same dosages and with administration routes as usedhereinbefore or about from 1 to 99% of the heretofore employed dosages.

In another embodiment of the invention, there is provided an article ofmanufacture containing materials useful for the treatment of osteolyticdisease, including cancer metastasis to bone and bone remodelingassociated with such diseases. The article of manufacture comprises acontainer and a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for treating the condition andmay have a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agent in the composition is theantibody of the invention. The label on, or associated with, thecontainer indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container containing a second therapeutic agent (including any ofthe second therapeutic agents for osteolytic diseases discussed hereinor known in the art). The article of manufacture may further compriseanother container containing a pharmaceutically-acceptable buffer, suchas phosphate-buffered saline, Ringer's solution or dextrose solution forreconstituting a lyophilized antibody formulation. It may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

Immunotherapy

Anti-M-CSF antibodies useful in treating patients having cancers includethose which are capable of initiating a potent immune response againstthe tumor and those which are capable of direct cytotoxicity. In thisregard, anti-M-CSF antibodies may elicit tumor cell lysis by eithercomplement-mediated or antibody-dependent cell cytotoxicity (ADCC)mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites or complement proteins. In addition, anti-M-CSF antibodies thatexert a direct biological effect on tumor growth are useful in thepractice of the invention. Potential mechanisms by which such directlycytotoxic antibodies may act include inhibition of cell growth,modulation of cellular differentiation, modulation of tumor angiogenesisfactor profiles, and the induction of apoptosis. The mechanism by whicha particular anti-M-CSF antibody exerts an anti-tumor effect may beevaluated using any number of in vitro assays designed to determineADCC, ADMMC, complement-mediated cell lysis, and so forth, as isgenerally known in the art.

In one embodiment, immunotherapy is carried out using antibodies thathave a higher affinity for the membrane-bound form of M-CSF (M-CSFα)than for the secreted forms of M-CSF. For example, antibodies may beprepared that specifically bind at or around the cleavage site of M-CSFαor to the portion of M-CSFα adjacent to the membrane. Such antibodiesmay also beneficially inhibit cleavage and release of the soluble activeportion of M-CSFα.

The anti-M-CSF antibodies may be administered in their “naked” orunconjugated form, or may have therapeutic agents conjugated to them. Inone embodiment, anti-M-CSF antibodies are used as a radiosensitizer. Insuch embodiments, the anti-M-CSF antibodies are conjugated to aradiosensitizing agent. The term “radiosensitizer,” as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Diseases that are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells.

The terms “electromagnetic radiation” and “radiation” as used hereininclude, but are not limited to, radiation having the wavelength of10⁻²⁰ to 100 meters. Preferred embodiments of the present inventionemploy the electromagnetic radiation of: gamma-radiation (10⁻²⁰ to 10⁻¹³m), X-ray radiation (10⁻¹² to 10⁻⁹ m), ultraviolet light (10 nm to 400nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0mm), and microwave radiation (1 mm to 30 cm).

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Many cancertreatment protocols currently employ radiosensitizers activated by theelectromagnetic radiation of X-rays. Examples of X-ray activatedradiosensitizers include, but are not limited to, the following:metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin(r), benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

In another embodiment, the antibody may be conjugated to a receptor(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a ligand (e.g., avidin) which is conjugatedto a cytotoxic agent (e.g., a radionuclide).

The present invention further provides the above-described antibodies indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescent or luminescent or bioluminescent labels(such as FITC or rhodamine, etc.), paramagnetic atoms, and the like.Procedures for accomplishing such labeling are well known in the art;for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem.18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval,E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth.13:215 (1976)).

“Label” refers to a detectable compound or composition which isconjugated directly or indirectly to the antibody. The label may itselfbe detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, may catalyze chemicalalteration of a substrate compound or composition which is detectable.Alternatively, the label may not be detectable on its own but may be anelement that is bound by another agent that is detectable (e.g. anepitope tag or one of a binding partner pair such as biotin-avidin,etc.) Thus, the antibody may comprise a label or tag that facilitatesits isolation, and methods of the invention to identify antibodiesinclude a step of isolating the M-CSF/antibody through interaction withthe label or tag.

Exemplary therapeutic immunoconjugates comprise the antibody describedherein conjugated to a cytotoxic agent such as a chemotherapeutic agent,toxin (e.g., an enzymatically active toxin of bacterial, fungal, plantor animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate). Fusion proteins are described in further detailbelow.

Production of immunconjugates is described in U.S. Pat. No. 6,306,393.Immunoconjugates can be prepared by indirectly conjugating a therapeuticagent to an antibody component. General techniques are described in Shihet al., Int. J. Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer46:1101-1106 (1990); and Shih et al., U.S. Pat. No. 5,057,313. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of drug,toxin, chelator, boron addends, or other therapeutic agent. Thisreaction results in an initial Schiff base (imine) linkage, which can bestabilized by reduction to a secondary amine to form the finalconjugate.

The carrier polymer is preferably an aminodextran or polypeptide of atleast 50 amino acid residues, although other substantially equivalentpolymer carriers can also be used. Preferably, the final immunoconjugateis soluble in an aqueous solution, such as mammalian serum, for ease ofadministration and effective targeting for use in therapy. Thus,solubilizing functions on the carrier polymer will enhance the serumsolubility of the final immunoconjugate. In particular, an aminodextranwill be preferred.

The process for preparing an immunoconjugate with an aminodextrancarrier typically begins with a dextran polymer, advantageously adextran of average molecular weight of about 10,000-100,000. The dextranis reacted with an oxidizing agent to affect a controlled oxidation of aportion of its carbohydrate rings to generate aldehyde groups. Theoxidation is conveniently effected with glycolytic chemical reagentssuch as NaIO₄, according to conventional procedures.

The oxidized dextran is then reacted with a polyamine, preferably adiamine, and more preferably, a mono- or polyhydroxy diamine. Suitableamines include ethylene diamine, propylene diamine, or other likepolymethylene diamines, diethylene triamine or like polyamines,1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups of the dextran is used to ensure substantially completeconversion of the aldehyde functions to Schiff base groups.

A reducing agent, such as NaBH₄, NaBH₃ CN or the like, is used to effectreductive stabilization of the resultant Schiff base intermediate. Theresultant adduct can be purified by passage through a conventionalsizing column to remove cross-linked dextrans.

Other conventional methods of derivatizing a dextran to introduce aminefunctions can also be used, e.g., reaction with cyanogen bromide,followed by reaction with a diamine.

The aminodextran is then reacted with a derivative of the particulardrug, toxin, chelator, immunomodulator, boron addend, or othertherapeutic agent to be loaded, in an activated form, preferably, acarboxyl-activated derivative, prepared by conventional means, e.g.,using dicyclohexylcarbodiimide (DCC) or a water soluble variant thereof,to form an intermediate adduct.

Alternatively, polypeptide toxins such as pokeweed antiviral protein orricin A-chain, and the like, can be coupled to aminodextran byglutaraldehyde condensation or by reaction of activated carboxyl groupson the protein with amines on the aminodextran.

Chelators for radiometals or magnetic resonance enhancers are well-knownin the art. Typical are derivatives of ethylenediaminetetraacetic acid(EDTA) and diethylenetriaminepentaacetic acid (DTPA). These chelatorstypically have groups on the side chain by which the chelator can beattached to a carrier. Such groups include, e.g., benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the amine group of acarrier. Alternatively, carboxyl groups or amine groups on a chelatorcan be coupled to a carrier by activation or prior derivatization andthen coupling, all by well-known means.

Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto antibody components to produce therapeutically usefulimmunoconjugates, as described below.

A polypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andimmunoconjugate.

Conjugation of the intermediate conjugate with the antibody component iseffected by oxidizing the carbohydrate portion of the antibody componentand reacting the resulting aldehyde (and ketone) carbonyls with aminegroups remaining on the carrier after loading with a drug, toxin,chelator, immunomodulator, boron addend, or other therapeutic agent.Alternatively, an intermediate conjugate can be attached to an oxidizedantibody component via amine groups that have been introduced in theintermediate conjugate after loading with the therapeutic agent.Oxidation is conveniently effected either chemically, e.g., with NaIO₄or other glycolytic reagent, or enzymatically, e.g., with neuraminidaseand galactose oxidase. In the case of an aminodextran carrier, not allof the amines of the aminodextran are typically used for loading atherapeutic agent. The remaining amines of aminodextran condense withthe oxidized antibody component to form Schiff base adducts, which arethen reductively stabilized, normally with a borohydride reducing agent.

Analogous procedures are used to produce other immunoconjugatesaccording to the invention. Loaded polypeptide carriers preferably havefree lysine residues remaining for condensation with the oxidizedcarbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamme.

The final immunoconjugate is purified using conventional techniques,such as sizing chromatography on Sephacryl S-300 or affinitychromatography using one or more CD84Hy epitopes.

Alternatively, immunoconjugates can be prepared by directly conjugatingan antibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

It will be appreciated that other therapeutic agents can be substitutedfor the chelators described herein. Those of skill in the art will beable to devise conjugation schemes without undue experimentation.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.Cancer56:244 (1994). General techniques for such conjugation arewell-known in the art. See, for example, Wong, Chemistry Of ProteinConjugation and Cross-Linking (CRC Press 1991); Upeslacis et al.,“Modification of Antibodies by Chemical Methods,” in MonoclonalAntibodies: Principles and Applications, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Enineering and Clinical Application, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to the antibody (see, e.g., WO94/11026).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion may be absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953. Theengineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a “divalent immunoconjugate” by attaching therapeutic agentsto a carbohydrate moiety and to a free sulfhydryl group. Such a freesulfhydryl group may be located in the hinge region of the antibodycomponent.

Anti-M-CSF Antibody Fusion Proteins

The present invention contemplates the use of fusion proteins comprisingone or more anti-M-CSF antibody moieties and another polypeptide, suchas an immunomodulator or toxin moiety. Methods of making antibody fusionproteins are well known in the art. See, e.g., U.S. Pat. No. 6,306,393.Antibody fusion proteins comprising an interleukin-2 moiety aredescribed by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al.,Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci.USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Huet al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum.Antibodies Hybridomas 6:129 (1995), describe a fusion protein thatincludes an F(ab′)₂ fragment and a tumor necrosis factor alpha moiety.

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch conjugates are ricin, abrin, ribonuclease, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.—A CancerJournal for Clinicians 44:43 (1994). Other suitable toxins are known tothose of skill in the art.

Antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of theinvention into free active drugs (See, e.g., Massey, Nature 328: 457-458(1987)). Antibody-abzyme conjugates can be prepared as described hereinfor delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312: 604-608. (1984))

Non-Therapeutic Uses

The antibodies of the invention may be used as affinity purificationagents for M-CSF or in diagnostic assays for M-CSF protein, e.g.,detecting its expression in specific cells, tissues, or serum. Theantibodies may also be used for in vivo diagnostic assays. Generally,for these purposes the antibody is labeled with a radionuclide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, such as ELISAs, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987). The antibodies may also be used for immunohistochemistry, tolabel tumor samples using methods known in the art.

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXAMPLES Example 1

This example shows that M-CSF antibodies RX1 and 5A1 are speciesspecific and that antibodies RX1, MC1, and MC3 neutralize human M-CSFactivity. RX1 is a commercially sold antibody that was available morethan a year prior to the filing date of this application. Exemplarycommercial sources include, but are not limited to, mouse anti-humanM-CSF monoclonal antibody clones 116, 692, and 21 (Anogen); anti-humanM-CSF antibody clones 21113.131, 26730, and 26786 (R & D Systems, Inc.);and anti-human M-CSF antibody clone M16 (Antigenix America, Inc.).

To test the neutralizing activity of RX1 and 5A1, a proliferation assayof M-NFS-60 cell line was used (American Type Culture CollectionAccession No. CRL-1838, available from ATCC in Rockville, Md., USA,derived from a myelogenous leukemia induced with the Cas-Br-MuLV wildmouse ecotropic retrovirous, responsive to both interleukin 3 and M-CSFand which contain a truncated c-myb proto-oncogene caused by theintegration of a retrovirus). Proliferation of M-NFS-60 requires activeM-CSF in a dose-dependent fashion. In the assay, M-NFS-60 cells werewashed and plated in RPMI 1640 medium with 10% FBS and 3000 U/ml ofM-CSF and 1% Pen/Strep. Recombinant human M-CSF (at 10 ng/ml finalconcentration), human or murine-specific, was incubated with variousconcentrations of antibodies for 1 hour at 37° C. in 5% CO₂ in anincubator. Following the incubation, the mixture was added to theM-NFS-60 culture in 96 well microtiter plates. The total assay volumeper well was 100 μl, with 10 ng/ml M-CSF, and the antibody concentrationindicated in FIG. 5. Cells were incubated at 37° C. under 5% CO₂ for 72hours before cell numbers were quantified by CellTiter Glo assay(Promega). The aforementioned assay was repeated for antibodies MC3 andMC1.

As shown in FIG. 5, M-CSF antibodies RX1 and 5A1 are species specific.Cell proliferation is presented as the fluorescent reading fromCellTiter Glo assay, which is linear with cell number. Species specificneutralizing activity of RX1 and 5A1 is shown by its ability to inhibitM-NFS-60 in the presence of either human or murine M-CSF. Finally, asshown in FIG. 5B, antibodies MC3 and MC1 are also effective inhibitorsof human M-CSF activity.

Example 2

This example shows that antibody RX1 effectively inhibits osteolysis ina human xenograft model at a dose of 5 mg/kg. Female nude mice at theage of 4-7 weeks old, average weight ˜20 g were used in this study.Tumor cells (MDA-MB-231, 3×10⁵) suspended in 10 μl of saline was beinjected into the right tibia bone marrow cavity. Radiograms of the hindlegs were taken one day after tumor inoculation for getting baselineimage and checking for bone fracture caused by injection. Mice wererandomized into treatment groups at 10 mice per group including PBS andRX1 at 5 mg/kg, injected i.p. once a week for 6 weeks. At the end ofstudy, radiograms of the hind legs were taken again and compared againstbaseline for bone damage. The degree of bone damage caused by tumor wasdefined as shown in FIG. 6. The group with RX1 5 mg/kg treatment showedstatistically significant protection of the bone from tumor-caseddamage.

Example 3

This example shows that the number of metastases is reduced whenantibody RX1 is administered to human breast cancer MDA-MB-231 bearingnude mice at a concentration of 5 mg/kg.

Female nude mice at the age of 4-7 weeks old, average weight ˜20 g wereused for this study. Tumor cells (MDA-MB-231, 3×10⁵) suspended in 10 μlof saline was injected into the right tibia bone marrow cavity.Radiograms of the hind legs were taken one day after tumor inoculationfor getting baseline image and checking for bone fracture caused byinjection. Mice were randomly grouped into the treatment groupsincluding PBS and RX1 at 5 mg/kg injected i.p. once a week for 6 weeks.At the end of study, lungs of each treatment group were collected andfixed in Bouin's solution for metastatic lung nodule counting.

As shown in FIG. 7, that the number of metastases is reduced whenantibody RX1 is administered to human breast cancer MDA-MB-231 bearingnude mice at a dose of 5 mg/kg.

Example 4

This example sets out a procedure for humanization of the RX1 antibody.5H4, MC1 and MC3 are humanized using similar procedures.

Design of Genes for Humanized RX1 Light and Heavy Chains

The nucleotide and amino acid sequence for murine RX1 are set forth inFIG. 4B. The sequence of a human antibody identified using the NationalBiomedical Foundation Protein Identification Resource or similardatabase is used to provide the framework of the humanized antibody. Toselect the sequence of the humanized heavy chain, the murine RX1 heavychain sequence is aligned with the sequence of the human antibody heavychain. At each position, the human antibody amino acid is selected forthe humanized sequence, unless that position falls in any one of fourcategories defined below, in which case the murine RX1 amino acid isselected:

(1) The position falls within a complementarity determining region(CDR), as defined by Kabat, J. Immunol., 125, 961-969 (1980);

(2) The human antibody amino acid is rare for human heavy chains at thatposition, whereas the murine RX1 amino acid is common for human heavychains at that position;

(3) The position is immediately adjacent to a CDR in the amino acidsequence of the murine RX1 heavy chain; or

(4) 3-dimensional modeling of the murine RX1 antibody suggests that theamino acid is physically close to the antigen binding region.

To select the sequence of the humanized light chain, the murine RX1light chain sequence is aligned with the sequence of the human antibodylight chain. The human antibody amino acid is selected at each positionfor the humanized sequence, unless the position again falls into one ofthe categories described above and repeated below:

(1) CDR's;

(2) murine RX1 amino acid more typical than human antibody;

(3) Adjacent to CDR's; or

(4) Possible 3-dimensional proximity to binding region.

The actual nucleotide sequence of the heavy and light chain genes isselected as follows:

(1) The nucleotide sequences code for the amino acid sequences chosen asdescribed above;

(2) 5′ of these coding sequences, the nucleotide sequences code for aleader

(signal) sequence. These leader sequences were chosen as typical ofantibodies;

(3) 3′ of the coding sequences, the nucleotide sequences are thesequences that follow the mouse light chain J5 segment and the mouseheavy chain J2 segment, which are part of the murine RX1 sequence. Thesesequences are included because they contain splice donor signals; and

(4) At each end of the sequence is an Xba I site to allow cutting at theXba I sites and cloning into the Xba I site of a vector.

Construction of Humanized Light and Heavy Chain Genes

To synthesize the heavy chain, four oligonucleotides are synthesizedusing an Applied Biosystems 380B DNA synthesizer. Two of theoligonucleotides are part of each strand of the heavy chain, and eacholigonucleotide overlaps the next one by about 20 nucleotides to allowannealing. Together, the oligonucleotides cover the entire humanizedheavy chain variable region with a few extra nucleotides at each end toallow cutting at the Xba I sites. The oligonucleotides are purified frompolyacrylamide gels.

Each oligonucleotide is phosphorylated using ATP and T4 polynucleotidekinase by standard procedures (Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)). To anneal the phosphorylated oligonucleotides,they are suspended together in 40 ul of TA (33 mM Tris acetate, pH 7.9,66 mM potassium acetate, 10 mM magnesium acetate) at a concentration ofabout 3.75 uM each, heated to 95° C. for 4 min. and cooled slowly to 4°C. To synthesize the complete gene from the oligonucleotides bysynthesizing the opposite strand of each oligonucleotide, the followingcomponents are added in a final volume of 100 ul:

10 ul annealed oligonucleotides 0.16 mM each deoxyribonucleotide 0.5 mMATP 0.5 mM DTT 100 ug/ml BSA 3.5 ug/ml T4 g43 protein (DNA polymerase)25 ug/ml T4 g44/62 protein (polymerase accessory protein) 25 ug/ml 45protein (polymerase accessory protein)

The mixture is incubated at 37° C. for 30 mM. Then 10 u of T4 DNA ligaseis added and incubation at 37° C. is resumed for 30 min. The polymeraseand ligase are inactivated by incubation of the reaction at 70° C. for15 min. To digest the gene with Xba I, 50 ul of 2×TA containing BSA at200 ug/ml and DTT at 1 mM, 43 ul of water, and 50 u of Xba I in 5 ul isadded to the reaction. The reaction is incubated for 3 hr at 37° C., andthen purified on a gel. The Xba I fragment is purified from a gel andcloned into the Xba I site of the plasmid pUC19 by standard methods.Plasmids are purified using standard techniques and sequenced using thedideoxy method.

Construction of plasmids to express humanized light and heavy chains isaccomplished by isolating the light and heavy chain Xba I fragments fromthe pUC19 plasmid in which it had been inserted and then inserting itinto the Xba I site of an appropriate expression vector which willexpress high levels of a complete heavy chain when transfected into anappropriate host cell.

Synthesis and Affinity of Humanized Antibody

The expression vectors are transfected into mouse Sp2/0 cells, and cellsthat integrate the plasmids are selected on the basis of the selectablemarker(s) conferred by the expression vectors by standard methods. Toverify that these cells secreted antibody that binds to M-CSF,supernatant from the cells are incubated with cells that are known toexpress M-CSF. After washing, the cells are incubated withfluorescein-conjugated goat anti-human antibody, washed, and analyzedfor fluorescence on a FACSCAN cytofluorometer.

For the next experiments, cells producing the humanized antibody areinjected into mice, and the resultant ascites is collected. Humanizedantibody is purified to substantial homogeneity from the ascites bypassage through an affinity column of goat anti-human immunoglobulinantibody, prepared on an Affigel-10 support (Bio-Rad Laboratories, Inc.,Richmond, Calif.) according to standard techniques. To determine theaffinity of the humanized antibody relative to the original murine RX1antibody, a competitive binding experiment is performed according totechniques known in the art.

Example 5

This example describes cloning and expression of Human Engineered™ RX1antibodies, as well as purification of such antibodies and testing forbinding activity. Human Engineered™ 5H4, MC1, and MC3 antibodies areprepared using similar procedures.

Design of Human Engineered™ Sequences

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position. Subtitutional changes arepreferred but insertions and deletions are also possible. FIGS. 4B and4C show the risk assignment for each amino acid residue of murine RX1light and heavy chains, respectively, categorized as a high, moderate orlow risk change.

Variable regions of the light and heavy chains of the murine RX1antibody were Human Engineered™ using this method. Amino acid residuesthat are candidates for modification according to the method at low riskpositions were identified by aligning the amino acid sequences of themurine variable regions with a human variable region sequence. Any humanvariable region can be used, including an individual VH or VL sequenceor a human consensus VH or VL sequence. The amino acid residues at anynumber of the low risk positions, or at all of the low risk positions,can be changed. For the Human Engineered™ “low risk” heavy chainsequence in FIGS. 19A-B, human consensus Vh2 (based on Kabat) was usedas the template, and for each position where the murine and human aminoacid residues differed at low risk positions, an amino acid modificationwas introduced that replaced the murine residue with the human residue.For the Human Engineered™ “low risk” light chain sequence in FIGS.20A-B, human consensus kappa 3 (based on Kabat) was used as thetemplate, and for each position where the murine and human amino acidresidues differed at low risk positions, an amino acid modification wasintroduced that replaced the murine residue with the human residue. Atotal of 16 amino acid low risk modifications were made to the lightchain and 8 low risk modifications were made to the heavy chain.

Similarly, amino acid residues that are candidates for modificationaccording to the method at all of the low and moderate risk positionswere identified by aligning the amino acid sequences of the murinevariable regions with a human variable region sequence. The amino acidresidues at any number of the low or moderate risk positions, or at allof the low and moderate risk positions, can be changed. For the HumanEngineered™ heavy chain sequence, in FIGS. 19A-B, human consensus Vh2(based on Kabat) was used as the template, and for each position wherethe murine and human amino acid residues differed at low or moderaterisk positions, an amino acid modification was introduced that replacedthe murine residue with the human residue. For the Human Engineered™light chain sequence in FIGS. 20A-B, human consensus kappa 3 (based onKabat) was used as the template, and for each position where the murineand human amino acid residues differed at low or moderate riskpositions, an amino acid modification was introduced that replaced themurine residue with the human residue. A total of 19 low and moderaterisk amino acid modifications were made to the light chain and 12 lowand moderate modifications were made to the heavy chain.

An “alternative low risk” light chain sequence was also prepared asshown in FIGS. 21A-B, in which the modification at position 54 wasreversed back to murine. An “alternative low+moderate risk” light chainsequence was also prepared as shown in FIGS. 21A-B, in which themodifications at positions 54-56 were reversed back to murine.

Finally, a Human Engineered™ “low+moderate risk” light chain V regionsequence also was produced using human germline VK6 subgroup 2-1-(1) A14as the template, as shown in FIGS. 22A-B.

Also contemplated by the present invention is retaining amino acids41-43 (NGS) of FIG. 4A which represent the glycosylation site.Alternatively, only one or two of amino acids 41-43 (e.g., NG) may beretained.

Preparation of Expression Vectors for Permanent Cell Line Development

DNA fragments encoding each of the above-described heavy and light chainV region sequences along with antibody-derived signal sequences wereconstructed using synthetic nucleotide synthesis. DNA encoding each ofthe light chain V region amino acid sequences described above wereinserted into vector pMXP10 containing the human Kappa light chainconstant region. DNA encoding each of the heavy chain V region aminoacid sequences described above were inserted into vector pMXP6containing the human Gamma-2 heavy chain constant region. Additionalvectors were constructed containing the heavy chain V region amino acidsequences fused to the human Gamma-1 (cDNA) and Gamma-4 (genomic andcDNA) constant regions having sequences displayed in FIGS. 29A, 29 b,and 30. All of these vectors contain a hCMV promoter and a mouse kappalight chain 3′ untranslated region as well as selectable marker genessuch as neo or or his for selection of G418 or histidinol—resistanttransfectants, respectively. The light and heavy chain vectors aredescribed in Tables 2 and 3, respectively.

TABLE 2 Single gene permanent Kappa light chain vectors. SelectivePlasmid V Region Marker pMXC5 Low + Mod Risk (Kabat) neo pMXC6 Low Risk(Kabat) neo pMXC13 Low Risk (Kabat) - R54 to S neo pMXC14 Low + Mod Risk(Kabat)-RAT54, 55, 56 to SIS neo pMXC22 Low + Mod Risk (Germline) neo

TABLE 3 Single gene permanent heavy chain vectors. Selective Plasmid VRegion C Region Marker pMXC7 Low + Mod Risk (Kabat) Gamma 2 neo pMXC8Low Risk (Kabat) Gamma 2 neo pMXC40 Low Risk (Kabat) Gamma 1 neo pMXC41Low + Mod Risk (Kabat) Gamma 1 neo pMXC45 Low + Mod Risk (Kabat) Gamma 4(genomic) neo pMXC46 Low + Mod Risk (Kabat) Gamma 4 (cDNA) neo

Vectors comprising the desired Human Engineered™ light plus heavy chaingenes (Gamma-1, Gamma-2 and Gamma-4) were then constructed. These“2-Gene” vectors contain genes encoding each antibody chain, heavy andlight, under control of the hCMV promoter, CMV splice donor, SV40 16Ssplice acceptor and the mouse kappa light chain 3′ untranslated DNAincluding the polyA site. They also contain a selectable marker genesuch as neo or his and the ampicillin resistance gene. Vectorscontaining both heavy and light chain genes are described in Table 4.Vectors comprising two copies of each light and heavy chain genes (fourgene vectors) also can be constructed.

TABLE 4 Two-gene permanent expression vectors Heavy Chain SelectivePlasmid Kappa Light Chain V region C region Marker pMXC12 Low Risk(Kabat) Low Risk (Kabat) Gamma 2 neo pMXC37 Low Risk (Kabat) Low Risk(Kabat) Gamma 2 his pMXC9 Low + Mod Risk (Kabat) Low + Mod Risk Gamma 2neo (Kabat) pMXC16 Low Risk (Kabat) Low + Mod Risk Gamma 2 neo (Kabat)pMXC17 Low + Mod Risk (Kabat) Low Risk (Kabat) Gamma 2 neo pMXC18 LowRisk (Kabat) R54 to S Low + Mod Risk Gamma 2 neo (Kabat) pMXC19 Low +Mod Risk Low + Mod Risk Gamma 2 neo (Kabat)-RAT54, 55, 56 to SIS (Kabat)pMXC20 Low Risk (Kabat) - R54 to S Low Risk (Kabat) Gamma 2 neo pMXC21Low + Mod Risk Low Risk (Kabat) Gamma 2 neo (Kabat)-RAT54, 55, 56 to SISpMXC25 Low + Mod Risk Low + Mod Risk Gamma 2 neo (Germline) (Kabat)pMXC47 Low + Mod Risk Low + Mod Risk Gamma 2 his (Germline) (Kabat)pMXC26 Low + Mod Risk Low Risk (Kabat) Gamma 2 neo (Germline) pMXC42Low + Mod Risk Low Risk (Kabat) Gamma 1 neo (Germline) pMXC43 Low + ModRisk Low + Mod Risk Gamma 1 neo (Germline) (Kabat) pMXC50 Low + Mod RiskLow + Mod Risk Gamma 1 his (Germline) (Kabat) pMXC48 Low + Mod RiskLow + Mod Risk Gamma 4 Neo (Germline) (Kabat) (cDNA) pMXC49 Low + ModRisk Low + Mod Risk Gamma 4 neo (Germline) (Kabat) (genomic)

Preparation of Expression Vectors for Transient Expression

Vectors containing either the light or heavy chain genes described abovealso were constructed for transient transfection. These vectors aresimilar to those described above for permanent transfections except thatinstead of the neo or his genes, they contain the Epstein-Barr virusoriP for replication in HEK293 cells that express the Epstein-Barr virusnuclear antigen. The vectors for transient transfection are described inTables 5 and 6.

TABLE 5 Transient Kappa light chain vectors. Plasmid V Region pMXC1Low + Mod Risk (Kabat) pMXC2 Low Risk (Kabat) pMXC10 Low + Mod Risk(Kabat)-RAT54, 55, 56 to SIS pMXC11 Low Risk (Kabat) - R54 to S pMXC15Low + Mod Risk (Germline)

TABLE 6 Transient heavy chain vectors. Plasmid V Region C Region pMXC3Low + Mod Risk (Kabat) Gamma 2 pMXC4 Low Risk (Kabat) Gamma 2 pMXC29 LowRisk (Kabat) Gamma 1 pMXC38 Low Risk (Kabat) Gamma 4 (genomic) pMXC39Low + Mod Risk (Kabat) Gamma 1

Transient Expression of Human-Engineered RX1 in HEK293E Cells

Separate vectors each containing oriP from the Epstein-Barr Virus andthe light chain or heavy chain genes described above were transfectedtransiently into HEK293E cells. Transiently transfected cells wereallowed to incubate for up to 10 days after which the supernatant wasrecovered and antibody purified using Protein A chromatography. Theproteins produced by transient transfection of 293E cells are describedin Table 7 below.

TABLE 7 Human-engineered RX1 antibodies prepared. Light Chain HeavyChain Antibody Plasmid Protein Plasmid Protein heRX1-1.G2 pMXC2 Low Risk(Kabat) pMXC4 Low Risk (Kabat) heRX1-2.G2 pMXC2 Low Risk (Kabat) pMXC3Low + Mod Risk (Kabat) heRX1-3.G2 pMXC1 Low + Mod Risk (Kabat) pMXC4 LowRisk (Kabat) heRX1-4.G2 pMXC1 Low + Mod Risk (Kabat) pMXC3 Low + ModRisk (Kabat) heRX1-5.G2 pMXC11 Low Risk (Kabat) - R54 to S pMXC4 LowRisk (Kabat) heRX1-6.G2 pMXC11 Low Risk (Kabat) - R54 to S pMXC4 LowRisk (Kabat) heRX1-7.G2 pMXC10 Low + Mod Risk pMXC4 Low Risk (Kabat)(Kabat)-RAT54, 55, 56 to SIS heRX1-8.G2 pMXC10 Low + Mod Risk pMXC3Low + Mod Risk (Kabat) (Kabat)-RAT 54, 55, 56 to SIS heRX1-9.G2 pMXC15Low + Mod Risk (Germline) pMXC4 Low Risk (Kabat) heRX1-10.G2 pMXC15Low + Mod Risk (Germline) pMXC3 Low + Mod Risk (Kabat) heRX1-1.G1 pMXC2Low Risk (Germline) pMXC29 Low Risk (Kabat) heRX1-10.G1 pMXC15 Low + ModRisk (Germline) pMXC39 Low + Mod Risk (Kabat) heRX1-9.G4 pMXC15 Low +Mod Risk (Germline) pMXC38 Low Risk (Kabat)

Development of Permanently Transfected CHO-K1 Cells

The vectors described above (Table 4) containing one copy each of thelight and heavy genes together are transfected into Ex-Cell 302-adaptedCHO-K1 cells. CHO-K¹ cells adapted to suspension growth in Ex-Cell 302medium are typically electroporated with 40 ug of linearized vector.Alternatively, linearized DNA can be complexed with linearpolyethyleneimine (PEI) and used for transfection. The cells are platedin 96 well plates containing Ex-Cell 302 medium supplemented with 1% FBSand G418. Clones are screened in 96 well plates and the top ˜10% ofclones from each transfection are transferred to 24 well platescontaining Ex-Cell 302 medium.

A productivity test is performed in 24 well plates in Ex-Cell 302 mediumfor cultures grown for 7 and 14 days at which time culture supernatantsare tested for levels of secreted antibody by an immunoglobulin ELISAassay for IgG.

The top clones are transferred to shake flasks containing Ex-Cell 302medium. As soon as the cells are adapted to suspension growth, a shakeflask test is performed with these clones in Ex-Cell 302 medium. Thecells are grown for up to 10 days in 125 ml Erlenmeyer flasks containing25 ml media. The flasks are opened at least every other day of theincubation period to allow for gas exchange and the levels ofimmunoglobulin polypeptide in the culture medium are determined by IgGELISA at the end of the incubation period. Multiple sequentialtransfections of the same cell line with two or three multi-unittranscription vectors results in clones and cell lines that exhibitfurther increases in levels of immunoglobulin production, preferably to300 μg/ml or more.

Purification

A process for the purification of immunoglobulin polypeptides fromvectors and all lines according to the invention may be designed.According to methods well known in the art, cells are removed byfiltration after termination. The filtrate is loaded onto a Protein Acolumn (in multiple passes, if needed). The column is washed and thenthe expressed and secreted immunoglobulin polypeptides are eluted fromthe column. For preparation of antibody product, the Protein A pool isheld at a low pH (pH 3 for a minimum of 30 minutes and a maximum of onehour) as a viral inactivation step. An adsorptive cation exchange stepis next used to further purify the product. The eluate from theadsorptive separation column is passed through a virus retaining filterto provide further clearance of potential viral particles. The filtrateis further purified by passing through an anion exchange column in whichthe product does not bind. Finally, the purification process isconcluded by transferring the product into the formulation bufferthrough diafiltration. The retentate is adjusted to a proteinconcentration of at least 1 mg/mL and a stabilizer is added.

Binding Activity

The MCSF binding activity of the recombinant Human Engineered™antibodies is evaluated. Protein is purified from shake flask culturesupernatants by passage over a protein A column followed byconcentration determination by A₂₈₀. Binding assays are performed asdescribed in Example 1 above or 13 below. Immulon II plates areprecoated with the sM-CSF antigen pre-diluted in a PBS coating solutionto immobilize it to the microplate. Various test concentrations of M-CSFranging from 0.25 to 20 ug/ml are added at 50 ul/well and incubated at4° C. overnight. The plates are then washed 3 times with PBS-0.05%Tween. Blocking is performed by adding in PBS-0.05% Tween 1% BSAfollowed by a 30 minute incubation at 37° C. Dilutions of immunoglobulinpolypeptides are prepared in PBS-0.05% Tween 1% BSA solution. 2- or3-fold serial dilutions are prepared and added (100 ul/well) induplicate or triplicate. After a 90 minute incubation at 37° C., themicroplate is washed 3 times with PBS-0.05% Tween. For signaldevelopment, goat anti-human IgG (gamma- or Fc-specific) secondaryantibody conjugated to peroxidase is added to each well and incubatedfor 60 minutes at 37° C. followed by addition of OPD at 0.4 mg/ml incitrate buffer plus 0.012% H₂O₂. After 5-10 minutes at room temperaturethe assay is stopped by the addition of 100 ul 1M H₂SO₄ and the platesare read at 490 nm. Both goat anti-human IgG (gamma-specific) and goatanti-human IgG (Fc-specific) antibodies have been employed.

Example 6

The following example sets out a procedure for the treatment of humansusing M-CSF-specific antibody, such as an RX1-derived or RX1-competingantibody, including an RX1 Human Engineered™ antibody with a modified orunmodified IgG1 or IgG4 constant region. The procedure can also befollowed for an MC1- or MC3-derived or MC1- or MC3-competing antibody.The expected efficacious dosing range is 2 ug/kg to 10 mg/kg. Thisestimation is based on following rationale substantiated by experimentaldata:

The measured M-CSF level in human plasma (both healthy and breast cancerpatients) is about 1 ng/ml. M-CSF neutralizing antibody RX1 has ameasured EC₅₀ of 2 ng/ml against 1 ng/ml human M-CSF. Accordingly, theeffective antibody concentration in human plasma is expected to be 10 to50,000 fold over its EC₅₀, i.e. 20 ng/ml to 100 ug/ml antibody in humanplasma. Based on PK studies, in order to effectuate this concentrationin human patients, a dosing of 2 μg/kg to 10 mg/kg is required to reach20 ng/ml to 100 ug/ml antibody concentration in plasma.

Example 7

This example sets out a procedure for the evaluation of the anti-canceractivity of anti-M-CSF monoclonal antibody in a subcutaneous model.Example 2 above showed that anti-M-CSF monoclonal antibody treatmentsignificantly inhibited the bone loss induced by tumor growth in bonemarrow. The purpose of this study is to evaluate whether the antibodycan also inhibit the tumor growth in soft tissue.

Female mice at the age of 10 weeks old, average weight ˜20 g will beused for this study. Mice will undergo an acclimation period of at least7 days prior to study start. On day 0, the right flank of nude mice willbe injected with Lewis lung carcinoma or CT26 cancer cellssubcutaneously at 5×10⁶ cells per mouse per 100 μl. When tumor volumereaches 100-200 mm³ (usually 1 week after tumor inoculation), mice willbe randomized into 5 groups at 10 mice per group as follows:

1) PBS

2) RX1

3) 5A1

4) mIgG1+rIgG1 isotype Ab control

5) 5A1+RX1

Mice will be treated intraperitoneally with the designated antibodies at10 mpk once a week for 4 weeks. When tumor volume reaches 2000 mm³, thestudy will be terminated. Alternatively, animals will also be euthanizedwhen any of the following situations are met: tumor surface ulcerationis bigger than 30% of total tumor surface area, significant body weightloss (>20%), dehydration, and moribund. Whole blood will be collectedfrom all of the mice and monocyte population will be analyzed as apotential surrogate marker. Measurements of tumor width and length willbe used to calculate tumor volume. It is expected that tumor growth willbe inhibted as a result of the foregoing experiment.

Example 8

The following example sets out a procedure for the evaluation ofcombination therapy for the treatment and prevention of severeosteolytic disease associated with cancer metastasis.

Experimental Design. The study described in Example 2 or 3 above isrepeated essentially as described with the following exceptions. Inaddition to the antibody or antibody combination set out in thetreatment groups below, the animals will receive one of the followingadditional treatments:

-   -   1. Bisphosphonate (e.g., Aredia; Zometa; Clodronate).    -   2. Surgery    -   3. Radiation    -   4. Chemotherapy    -   5. Hormone therapy (e.g., Tamoxifen; anti-Androgen therapy)    -   6. Antibody therapy (e.g., RANKL/RANK neutralizing antibodies;        PTHrP neutralizing antibody)    -   7. Therapeutic protein therapy (e.g., soluble RANKL receptor;        OPG, and PDGF and MMP inhibitors)    -   8. Small molecule drug therapy (e.g., Src-kinase inhibitor)    -   9. Oligonucleotides therapy (e.g., RANKL or RANK or PTHrP        Anti-sense)    -   10. Gene therapy (e.g., RANKL or RANK inhibitors)    -   11. Peptide therapy (e.g. muteins of RANKL)

The treatment groups are as follows. The above additional treatments areindicated below as “plus therapy X”:

-   -   1. PBS only    -   2. treatment with therapy X only    -   3. rat IgG1 isotype control    -   4. murine IG1 isotype control    -   5. RX1 anti-human MCSF only    -   6. 5A1 rat IgG1 anti-mouse MCSF only    -   7. rat IgG1 and murine IgG1 isotype control combination    -   8. RX1 an 5A1 combination    -   9. rat IgG1 isotype control plus therapy X    -   10. murine IG1 isotype control plus therapy X    -   11. RX1 anti-human MCSF plus therapy X    -   12. 5A1 rat IgG1 anti-mouse MCSF plus therapy X    -   13. rat IgG1 and murine IgG1 isotype control combination plus        therapy X    -   14. RX1 and 5A1 combination plus therapy X

Dosing: 0.1-30 mg/kg each antibody is used for administration to eachanimal. Preferred dosing is 10 mg/kg. The administration route can beIV, IP, SC. The preferred route is IP. Treatment will begin the dayfollowing injection of tumor cells, as described in Example 6, above.

Measurements. To assess the severity of osteolysis among the varioustreatment groups, each mouse receives a baseline Faxitron image takenthe day following injection of tumor cells. A Faxitron image is alsotaken at the end of the study (8 weeks). Tumor growth is simultaneouslymeasured using the Xenogen system since the tumor cells stably expressluciferase. It is expected that combination therapy for the treatmentand prevention severe osteolytic disease associated with cancermetastasis will be improved with relative to antibody therapy alone.

Example 9

The following example provides a protocol for evaluating the ability ofM-CSF-specific antibody to bind to, for example, breast cancer cells(cell line MDA231) or multiple myeloma cancer cells (cell line ARH77)using a fluorescence-activated cell sorter.

The cells were first washed twice with PBS (no Ca²⁺, Mg²⁺). For each10-cm plate, 2 ml of 3 mM EDTA was added, and the paltes were incubatedat 37° C. for 2-3 minutes, until the cells were rounded and began todetach from the dish. Next, 10 ml of buffer A (PBS+5% FBS) was added andmixed. At that time, the cells were pelleted and resuspended at about5×10⁶ cells/ml in PBS+5% FBS, and the cells were placed into tubules at100 μL/sample.

At this point, 0.1-10 ug/ml of the primary antibody (used at indicatedconcentration of M-CSF antibody or control antibody) was added.Dilution, if necessary, was made in 5% FBS/PBS. The mixture was thenincubated for 30 min at 4° C. Following the incubation period, the cellswere washed 3 times by centrifugation at 400 g for 5 min., and the cellswere resuspended in PBS.

The FITC or PE-labeld anti-IgG antibody (0.25 ug/sample) was diluted in1% BSA/PBS at the optimal dilution, and the cells were resuspended inthis solution and incubated for 30 min at 4° C. Next, the cells werewashed 3 times as described above. Following th cell washes, the cellswere resuspended with 0.5 ml/sample PI-PBS (if necessary to distinguishdead cells from live ones). The cells can also be fixed for lateranalysis (the cells can last about 3 days if they are fixed with 0.1%formaldehyde). The cells were next analyzed in a fluorescence-activeFACS using standard procedures.

As shown in FIGS. 8A and 8B, an MCSF-specific antibody RX1 bound tobreast cancer cell line MDA231 or to multiple myeloma cancer cell lineARH77 at a variety of antibody concentrations as indicated.

Example 10

The following example shows M-CSF is prevalent on a number of cancercell surfaces Immunohistochemical staining of M-CSF was carried outusing a M-CSF-specific antibody RX1 as follows.

At the outset, slides were heated in anoven at 55-60° C. for 1 hour andallowed to cool for 2-3 minutes. The following de-waxing andre-hydration parameters were used:

a. Xylene 3 × 5 minutes b. 100% Reagent Alcohol 2 × 5 minutes c. 95%Reagent Alcohol 2 × 4 minutes d. 75% Reagent Alcohol 2 × 3 minutes e.50% Reagent Alcohol 1 × 3 minutes g. dI H2O 2-3 quick rinses

Prior to the peroxide blocking step, antigen retrieval was preparedusing 1× Biogenex Citra Plus. The solution was initially microwaved atfull power to boil. Once the solution boiled, the microwave was quicklyset for another 13 min at power-level 2, and allowed to cool beforeproceeding. The peroxide blocking step was performed as follows. Theslides were immersed slides in 3% H₂O₂ (25 ml 30% to 250 ml dI H₂O) andplaced at room temperature for 10 minutes. The slides were next rinsed2× with dI H₂O, and washed with 1×PBS 2×2 minutes.

The avidin/biotin blocking procedure was performed as follows. Slideswere palced flat on a metal rack. A Blue PAP pen was used (hydrophobicslide marker) around tissue. Next, 2 drops Zymed Avidin (ReagentA)—enough to cover tissue—was added and the slides were incuabated atroom temperature for 10 min. Following the incubation, the slides werewashed as follows:

2×3 minute washes in 1×PBS.

2 drops Zymed Biotin (Reagent B), room temperature for 10 min. 2×3minute washes in 1×PBS.

The protein blocking procedure was performed as follows. First, 10%serum [to 2% final concentration] of secondary antibody species wasadded. The BioGenex Power Block was next diluted to 1× with dI H₂O. Therack of slides was immersed in Power Block for 8 min at roomtemperature, and the slides were rinsed 2× in 1×PBS.

For the addition of the primary antibody (RX1), the slides were placedflat on a metal rack. Antibody was added to cover each section (˜350μl), and the antibody was spread with pipet tip (if necessary) withoutscraping tissue. The slides were then incubated for 1 hour at roomtemperature. Following the incubation, the slides were washed 3× with1×PBS 3-5 minutes each time. At this point, BioGenex Multi-Link wasapplied to sections & incubated for 10-11 minutes at room temperature.The sections were then washed 3 minutes each time.

Labelling was performed by applying BioGenex HRP Label to sections,which were then incubated at room temperature for 10-11 min and washedwith 1×PBS 3×3 minutes. Next, BioGenex H₂O₂ substrate was added (1 dropAEC for every 2.5 ml H₂O₂) to the sections and incubated at roomtemperature for 10 min. The sections were then rinsed several times withdI H₂O. The counterstaining step was performed as follows. The sectionswere staine with hematoxylin for 1 minute at room temperature. Next, thesections were rinse with H₂O twice, and then incubated in 1×PBS for 1minute. Sections were then rinsed well with H₂O to remove PBS. Sectionswere mounted by applying a drop of BioGenex Super Mount to the sectionsection and then air drying over night at room temperature.

As shown in FIG. 9, M-CSF is prevalent on a number of cancer cellsurfaces. Sections for the indicated cancer cell types were scored asfollows:

-   -   0 No staining    -   1 Staining was similar to background    -   2 Positive, but weak staining    -   3 Positive and significant staining    -   4 Positive and strong staining

Example 11

The following example shows the procedure for producing antibodies MC1and MC3. MC1 and MC3 are two monoclonal murine antibodies thatneutralize human M-CSF antibody and bind to human M-CSF. The amino acidsequences of these antibodies are shown in FIGS. 14 and 15,respectively. They were identified by a series of steps including a)immunization of Balb C mice with recombinant human M-CSF; b) screeningfor positive clones that produce antibodies which bind to human M-CSF inan ELISA format; c) subcloning of positive clones to generate stablehybridoma clones; d) scale-up of cell culture to produce large quantityof antibodies; e) purification and characterization of antibodies inaffinity analysis, cell binding, and neutralizing activity assay asdescribed in previous examples.

FIGS. 16A and 16B show the alignment of the CDRs of the heavy and lightchains, respectively, of antibodies RX1, 5H4, MC1 and MC3.

Humanized and Human Engineered™ versions are generated as described inthe examples above.

Example 12

This example shows that M-CSF antibodies RX1 and 5H4, as well as Fabfragments thereof, have different neutralizing activities. The fallowingexample also shows that antibodies RX1, 5H4, and MC3 have varyingaffinities for M-CSF. This example further demonstrates that theaffinities of the aforementioned intact antibodies are higher relativeto Fab fragments of the aforementioned antibodies.

Neutralization activities of intact RX1 and 5H4 versus Fab fragments ofRX1 and 5H4 were determined by measuring M-CSF-dependent cellproliferation in the presence of various concentrations of antibody. Thecell proliferation was determined by chemiluminescent dye. As shown inFIG. 17, intact RX1 has the highest potency, while the Fab fragment ofRX1 loses its potency and behaves like 5H4 and the 5H4Fab fragment.

Binding properties of the aforementioned antibodies were analyzed usingBiacore analyses. In order to determine the relative affinities of RX1,5H4, and MC3 to M-CSF, rabbit anti-mouse Fc was immobilized onto a CM5biosensor chip via amine coupling. The aforementioned antibodies werethen captured on the anti-mouse Fc/CM5 biosensor chip at 1.5 μg/ml for 3mM at 2 μl/min. MCSF was flowed over the modified biosensor surface atvarying concentrations (Rmax ˜15). Test antibodies and antigen werediluted in 0.01 M HEPES pH 7.4, 0.15 M NaCL, 3 mM EDTA, 0.005%Surfactant P20 (HBS-EP). All experiments were performed at 25° C.Kinetic and affinity constants were determined using Biaevaluationsoftware (Biacore) with a 1:1 interaction model/global fit. As shownbelow in Table 8, RX1 binds to M-CSF with the highest affinity relativeto 5H4 and MC3.

TABLE 8 Ka (M−1 Sec−1) Kd (sec−1) KD (nM) RX1 1.64e6 2.7e−4 0.16 5H45.94e5 1.77e−3 3.0 MC3 7.04e5 1.93e−4 0.27

To determine the relative differences in the binding affinity of intactMab and Fab fragments of RX1, 5H4, and MC3, an alternate configurationwas used in the Biacore analysis. Specifically, M-CSF was immobilizedonto CM5 biosensor chip via amine coupling. 0.05 μg/ml M-CSF in 10 mM NaAcetate pH 4.0 was injected at 1 μl/min for 5 minutes to achieve RL=6-12RU. Test antibody (or Fab fragment) were flowed over the modifiedbiosensor surface at varying concentrations. Test antibodies werediluted in 0.01 M HEPES pH 7.4, 0.15 M NaCL, 3 mM EDTA, 0.005%Surfactant P20 (HBS-EP) and all experiments were done at 25° C. Kineticand affinity constants were determined using Biaevaluation software(Biacore) with a 1:1 interaction model/global fit. As shown below inTable 9, RX1 binds M-CSF with the highest affinity relative to the otherantibodies tested. The Fab fragment of RX1 binds M-SCF with asignificantly lower affinity relative to the RX1 holoprotein.

TABLE 9 Ka (M−1 Sec−1) Kd (sec−1) KD (nM) rRX1(mouse) 2.34e5 2.35e−4 1.0rRX1 Fab (mouse) 2.81e5 3.03e−3 10.8 5H4 1.27e5 1.26e−3 9.9 5H4 Fab2.04e5 2.85e−3 14.0

The binding, affinity and neutralization data indicate that theneutralization activity of RX1 is due primarily to its remarkably highaffinity for M-CSF, and that this high affinity may be due at least inpart to the ability of both amus of the antibody to bind the M-CSF dimersimultaneously.

Example 13

The following example reveals the linear epitope (i.e., amino acidsequence) on M-CSF recognized by antibodies RX1, 5H4, and MC3.

Initially, the epitope mapping strategy was designed to determinewhether antibodies RX1, 5H4, and MC3 recognized linear epitopes orconformational epitopes within M-CSF. Accordingly, the anti-M-CSFantibodies were tested against 0.1 μM-CSF under reducing as well asnon-reducing conditions. Only the non-reduced form of M-CSF wasrecognized by each of the antibodies, suggesting the epitopes recognizedare discontinuous in nature.

Next, the linear epitope of M-CSF was determined for each antibody.Specifically, SPOTs membranes (Sigma Genosys) were prepared where theM-CSF fragment sequence of interest, overlapping 10mer peptidessynthesized with one amino acid offset, were loaded onto the cellulosemembrane support. These membranes were then probed with theaforementioned antibodies and reactive SPOTs were identified. Thepeptide sequence was then identified by its corresponding location onthe membrane, and overlapping amino acids within the positive reactingpeptides were identified as the epitope. As shown in FIG. 18, RX1 bindsto a different linear epitope than 5H4 and MC3, which map to a differentlocation on M-CSF. RX1 binds to a linear epitope represented by RFRDNTPN(SEQ ID NO: 120) or RFRDNTAN (SEQ ID NO: 121), amino acids 98-105 ofM-CSF of FIG. 12. 5H4 binds to a linear epitope represented by ITFEFVDQE(SEQ ID NO: 122), amino acids 65-73 of M-CSF of FIG. 12. MC3 binds totwo linear epitopes represented by (1) ITFEFVDQE (SEQ ID NO: 122), aminoacids 65-73 of M-CSF of FIGS. 12 and (2) FYETPLQ (SEQ ID NO: 123), aminoacids 138-144 of M-CSF of FIG. 12.

Example 14

The binding affinity of the Human Engineered™ versions of RX1 antibodiesprepared as described above in Example 5 was determined. This exampleshows that Human Engineered™ RX1 antibodies with different IgG subclassconstant regions bind M-CSF with different affinities in vitro. Todetermine the relative differences in the binding affinity of intactantibodies by Biacore analysis, M-CSF was immobilized onto CM5 biosensorchip via amine coupling. 0.05 μg/ml M-CSF in 10 mM Na-Acetate pH 4.0 wasinjected at 1 μl/min for 5 minutes to achieve RL=6-12 RU. Test antibodyor Fab fragments were flowed over the modified biosensor surface atvarying concentrations ranging from 100 nM to 1.5 nM in 2-folddilutions. Test antibodies were diluted in 0.01 M HEPES pH 7.4, 0.15 MNaCL, 3 mM EDTA, 0.005% Surfactant P20 (HBS-EP) and all experiments weredone at 25° C. Each concentration point and buffer blanks were run intriplicate, and data was collected over 3 minutes of association and 8minutes of dissociation. Kinetic and affinity constants were determinedusing Biaevaluation software with a 1:1 interaction model/global fit. Asshown in Table 10 below, heRX1-1.G1 and heRX1-1.G4 binds M-CSF withaffinities that most closely resemble the murine RX1M-CSF bindingaffinity.

TABLE 10 Antibody ka (M−1 sec−1) kd (sec−1) KD (nM) Murine RX1 (2.23 ±0.35) × 10⁵ (1.56 ± 0.67) × 10⁻⁴  0.7 ± 0.27 n = 21 heRX1-1.G2 (2.36 ±0.18) × 10⁵ (1.37 ± 0.24) × 10⁻³ 5.9 ± 1.4 n = 5 heRX1-10.G2 (1.73 ±0.29) × 10⁵  (1.1 ± 0.11) × 10⁻³ 6.3 ± 1.7 n = 2 heRX1-1.G1 2.50 × 10⁵2.38 × 10⁻⁴ 0.95 heRX1-1.G4 2.07 × 10⁵ 2.93 × 10⁻⁴ 1.42

In contrast, as shown in Table 11 below, although there was somevariation in binding affinity, all of the Gamma-2 constructs displayedat least a 7-fold decrease in binding affinity compared to the parentmurine antibody.

TABLE 11 Antibody ka (M−1 sec−1) kd (sec−1) KD (nM) Murine RX1 (2.23 ±0.35) × 10⁵ (1.56 ± 0.67) × 10⁻⁴  0.7 ± 0.27 n = 21 heRX1-1.G2 (2.36 ±0.18) × 10⁵ (1.37 ± 0.24) × 10⁻³ 5.9 ± 1.4 n = 5 heRX1-2.G2 2.18 × 10⁵1.65 × 10⁻³ 7.6 heRX1-3.G2 2.01 × 10⁵ 1.18 × 10⁻³ 5.9 heRX1-4.G2 2.38 ×10⁵ 1.08 × 10⁻³ 4.6 heRX1-5.G2 1.75 × 10⁵ 1.29 × 10⁻³ 7.4 heRX1-6.G21.88 × 10⁵ 1.49 × 10⁻³ 7.9 heRX1-7.G2 1.57 × 10⁵ 1.49 × 10⁻³ 9.5heRX1-8.G2 1.52 × 10⁵ 1.48 × 10⁻³ 9.8 heRX1-9.G2   2 × 10⁵ 1.44 × 10⁻³7.2 heRX1-10.G2 (1.73 ± 0.29) × 10⁵  (1.1 + 0.11) × 10⁻³ 6.3 ± 1.7 n = 2

Example 15

This example shows that Human Engineered™ RX1 antibodies with differentIgG subclass constant regions possess different neutralizationactivities in vitro. To test the neutralizing activity of M-CSFantibodies, a proliferation assay of M-NFS-60 cell line was used(American Type Culture Collection, Accession No. CRL-1838, availablefrom ATCC in Rockville, Md., USA, derived from a myologenous leukemiainduced with the Cas-Br.MuLV wild mouse ecotropic retrovirus. The cellline responds to both interleukin 3 and M-CSF and contains a truncatedc-myb proto-oncogene caused by the integration of a retrovirus).Proliferation of M-NFS-60 requires active M-CSF in a dose-dependentfashion. In the assay, M-NFS-60 cells were washed and plated in RPIM1640medium with 10% FBS and 1% Pen/Strep. Recombinant human M-CSF (at 10ng/ml final concentration, which is equivalent to 3000 U/ml of M-CSFactivity), was incubated with various concentrations of antibodiesranging from 1 ug/ml to 0.5 ng/ml (in serial 2-fold dilution) for 1 hourat 37° C. in 5% CO₂ in an incubator. Following the incubation, themixture was added to the M-NFS-60 culture in 96 well microtiter plates.The total assay volume per well was 100 ul, with 10 ng/ml M-CSF, and theantibody concentration indicated. Cells were incubated at 37° C. under5% CO₂ for 72 hours before cell numbers were quantified by CellTiter Gloassay (Promega). Each antibody was tested in triplicate, with a repeaton the following day (for a total of six assays per antibody). The IC50of each antibody was analyzed by curve fit.

The assay was repeated using human MCSF in serum, MDA231 conditionedmedium (which contains M-CSF), cynomologous monkey MCSF in serum, andcynomologous monkey recombinant MCSF. The results are presented in FIGS.25 (recombinant MCSF), 26 (human MCSF in serum) and 27 (MDA231conditioned medium) as the fluorescent reading from CellTiter Glo assay,which is linear with cell number. Neutralizing activity of theantibodies is shown as an inhibition of the proliferation of M-NFS-60cells.

The results show that the IC50 of heRX1-1-IgG1 and heRX1-1-IgG4 wereabout the same as the recombinant murine parent RX1 antibody, while theIC50 of heRX1-1-IgG2 was about 2-fold to 4-fold higher.

Table 12 below shows the relative IC50 (in terms of IC50 fold loss) ofthe various IgG2 constructs prepared as described above in Example 5. Ofthese constructs, heRX1-1.G2 and heRX1-10.G2 showed the least reductionin IC50.

TABLE 12 IC50 Antibody Fold Loss heRX1-1.G2 2.8× heRX1-2.G2 3.1×heRX1-3.G2 5.3× heRX1-4.G2 4.6× heRX1-5.G2 6.5× heRX1-6.G2 5.9×heRX1-7.G2 6.1× heRX1-8.G2 5.9× heRX1-9.G2 3.6× heRX1-10.G2 2.2×heRX1-1.G1 No loss heRX1-1.G4 No loss heRX1-10.G1 No loss heRX1-10.G4 Noloss

Example 16

This example shows that Human Engineered™ RX1 antibodies with differentIgG subclass constant regions result in different TRAP activities in anin vitro osteoclastogenesis assay.

The human bone marrow CD34+ cells (Biowhittaker catalog number 2M-101A,3×10⁵ cells/vial) were induced to differentiate into osteoclasts underthe experimental conditions described herein. On Day 1, CD34+ cells werethawed from one frozen vial into 10 ml of media (Alpha MEM with 10% FCS,1× Pen Strep and 1× fungizone). The cells were washed once andre-suspended in 2 ml of media and placed into a 96 well plate at 100 ulper well. On Day 2, without removing the original media, 50 ul of4×CSF-1 to 30 ng/ml final concentration and 50 ul of 4×RANKL (sRANKL,Chemicon catalog #GF091, 10 ug/package) to final concentration of 100ng/ml was added to each well. On Day 7, 50 ul of 5×RANKL to finalconcentration of 100 ng/ml was added to each well. On Day 17, the cellswere stained for TRAP activity (Leukocyte Acid Phosphatase kit for TRAPstaining, Sigma catalog# 387-A) and examined under the microscope.

M-CSF-neutralizing antibodies are added on Day 2 of the assay. Theantibodies inhibit osteoclast differentiation in a dose-dependent manneras shown in FIG. 28. The inhibitory activity of the antibodies in theosteoclast differentiation assay is shown as lack of visible osteoclastson Day 17 of the assay.

Example 17

The Human Engineered™ RX1 antibodies with different IgG subclassconstant regions were further characterized.

The antigen-antibody complexes that the various Human Engineered™ RX1antibodies formed with MCSF were studied by combining M-CSF and antibodyin buffer at equal molar ratios, followed by analysis of the size of theantigen-antibody complex using size exclusion chromatography or lightscattering. The results showed that the murine parent RX1 appeared toform 1:1 complexes with MCSF of about 200 kDa. The heRX1-9.G2 or 1-10.G2antibodies appeared to form 2:1 or 2:2 complexes with MCSF of about 400kDa. The heRX1-1.G2 appeared to form large lattice aggregates with MCSFof greater than 2×10⁶ Da. The IgG1 and IgG4 constructs formed smallcomplexes similar to that of murine parent RX1.

Denaturing reducing and non-reducing SDS-PAGE of heRX1-1.G4 showed thatthe IgG4 version appeared to form half-antibodies, as expected.

Example 18

This example shows the effect of Human Engineered™ RX1 antibody or otheranti-M-CSF antibodies on osteoclastogenesis in vitro. Primary humanOsteoclast Precursors (Cambrex Bio Science Walkersville, Inc. Cat#2T-110) are seeded at 10,000 cells per well at 0.2 ml/well in the cellculture media containing 30 ng/ml human M-CSF and 100 ng/ml RANKL. Testanti-M-CSF antibodies are added to the wells at indicated concentrationson the same day of cell plating. The cells are incubated at 37° C., in ahumidified atmosphere of 5% CO₂. On day 7, osteoclasts can be identifiedby phase microscopy as unusually large multinucleate cells. The culturecan be continued for an additional week, with feeding, during which timethe osteoclasts will continue to increase in size. At the end of theculture, cells are Stained for tartrate-resistant acid phosphatase, thepositive staining of which indicates the presence of osteoclasts. Thenumber of osteoclasts in each well is identified and counted under thelight microscope and can be shown in a graph as an average ofosteoclasts under the viewing field of the microscope.

Results of testing murine anti-MCSF antibody 5H4 are shown in FIG. 31.

Example 19

The following example sets forth an intratibial model to assess theability of anti-M-CSF antibodies to reduce the effects of mixedosteoblastic/osteoclastic bone lesions.

Male SCID mice, aged 4-5 weeks, average weight 15-18 g are used. Miceare chipped for identification, and undergo a 7 day acclimation periodprior to the study start date. Mice will receive s.c. injection offlunixin (2.5 mpk) 30 minutes before cell implantation. Mice are nextanesthetized via isoflurane inhalation; their right hind leg cleanedwith 70% ethanol and tumor cells (2.5×10⁵/10 ul saline) injected intothe right tibia bone marrow cavity using a 50 or 100 ml micro-syringe. Atotal of 20 mice are inoculated.

One day following tumor cell injection, the mice are imaged by aFaxitron machine to establish baseline measurements and to ensure thatthe injections did not damage the bone. Treatment groups (treatment willbegin day 2) are as follows:

-   -   Murine/Human IgG isotype control: 10 mpk each, i.p. once a week        for 5 weeks    -   Anti-MCSF (rat anti-mouse IgG1 5A1 and mouse anti-human IgG1        5H4): 10 mpk each, i.p. once a week for 5 weeks

Bone damage (assessed by Faxitron analysis once/week from week 1 untilbone damage in the first mouse of the control group reaches stage 3, atwhich time Faxitron analysis is performed twice a week) is monitored andbody weight is taken on a weekly basis. Mice are observed for clinicalsigns of distress daily until termination of study. Mice that developsevere clinical signs (>15% weight loss, hunched posture, inability toput weight on right leg, immobility, cold to the touch, leg swelling) orreaching stage 4 disease will be euthanized. At the completion of thestudy, final imaging, blood collection (heparanized) by cardiacpuncture. from all 20 animals in the study, and tibia collection (3right tibias from each group for histology, with 3 normal-left sidetibias) is performed.

Example 20

This example sets out an in vivo procedure for assessing periprostheticbone loss in the presence of anti-M-CSF antibodies. The generation andbiological effects of wear debris, especially polyethylene particles,are a major concern to surgeons, due to effects on the longevity oftotal joint replacements. Particulate debris induce a number of factorsthat interfere with normal homeostatic mechanisms of bone by promotingosteoclast differentiation and maturation, and inhibiting osteoblastfunction. The net effect is bone loss, undermining the periprostheticbone bed (Goodman et al., J. Biomed. Mater. Res., 65A: 454-461 (2003)).In this procedure, a drug test chamber (DTC) is surgically implanted inthe bone within a rabbit's limb and allowed to integrate within thebone. Subsequently, polyethylene particles, with or without drug, areinfused into the cavity, and bone tissue is periodically removed toassess bone formation.

The drug test chamber (DTC)

The DTC is a titanium device that is implanted at the level of thecortex in the proximal medial tibial metaphysis of rabbits, similar tothe bone harvest chamber. The removable core of the DTC is provided witha 10-μL cavity acting as a fluid reservoir linked to the 1×1×5-mmingrowth canal via a 1-mm long 0.25-mm diameter diffusion capillary. Forcontinuous infusion, two tubes are connected to the reservoir. One tubeis connected to a subcutaneous osmotic minipump (Alza Corp, Palo Alto,Calif.). The other tube is open to the subcutis, thereby functioning asa drain for the reservoir. This allows a slow continuous flow of thesolution through the reservoir, creating a concentration gradient alongthe diffusion capillary. The percolation of the solvent through thediffusion capillary to the bone is minimal due to the reservoir beingdrained to the subcutaneous tissue. Thus, the ingrowing tissue can belocally treated with a solution without disturbance of solvent flow.After each treatment, through a small 1-cm incision, the chamber isexposed and disassembled, leaving the outer osseointegrated cylinderintact within bone. The inner core is temporarily removed, exposing thetissue in the chamber, which is harvested for subsequent analysis. Thechamber parts are then reassembled for the next treatment. At the sametime, through another small incision, the minipump and tubing(containing the new solution to be infused) are replaced.

Surgical Procedure

DTCs are first implanted in mature, male, New Zealand white rabbits,preferably aged 6-12 months and weighing 3.5-4.5 kg under generalendotracheal anesthesia, as has been previously described. One dose ofcephalosporin antibiotic is given as prophylaxis for infection. After a6-week period for osseointegration, the contents of the chamber areharvested and discarded. The DTC is then connected, via polyvinyltubing, to an Alzet osmotic diffusion pump (Model # 2004, Alza, PaloAlto, Calif.). The diffusion pump contains 200 μL of fluid, and has adelivery rate of 0.25 μL/h. Carrier solution alone (i.e., theformulation solution without M-CSF antibodies, diluent, heparinsulphate, BSA, and the like) is infused for 3 weeks, followed by aharvest.

All rabbits are given M-CSF antibody treatments sequentially (see TableI). Anti-M-CSF antibody is first infused at dosages of 0, 0.5, 5, 50, or500 ng/day for 3-week periods in the absence of polyethylene particles.The contents of the chamber are harvested after each treatment, and thediffusion pump and tubing are exchanged for another one with theappropriate solution. Ultra high molecular weight polyethylene particlesat either a low dose (5.8×10¹¹ particles/mL) or high dose (1.7×10¹²particles/mL) are then placed in the DTC, with and without infusion of50 ng/day of anti-M-CSF antibody for 3 weeks. The contents of thechamber are harvested after each treatment and a new diffusion pump, andtubing with the appropriate solution, are placed in the rabbit.

Tissue Processing and Immunohistochemistry.

Harvested specimens are placed in optimal cutting medium (OCT), snapfrozen in liquid nitrogen, and stored at −70° C. until processed. Serial6 μm sections are cut using a cryostat (Cambridge Instruments, Buffalo,N.Y.) and mounted on microscope slides. Five or more sections from thecenter of the specimen are stained with hematoxylin and eosin forgeneral morphological characterization. Sequential sections on bothsides of the central sections above are used for immunoperoxidasestaining using monoclonal antibodies to identify cells expressing thevitronectin receptor (osteoclast-like cells).

Frozen sections are fixed in absolute acetone at 4° C. The slides arefixed in the acetone for 10 min, air dried, and treated with the acetonefor an additional 10 min. The slides are then dried for 45 min, placedin an airtight container, and stored at −70° C. until processed. Afterthawing, the sections are incubated at room temperature with monoclonalantibodies to identify osteoclast-like cells exhibiting the vitronectinreceptor (CD51, mouse antihuman monoclonal antibody to integrin αVβ3,Chemicon, Temecula, Calif.). The use of this monoclonal antibody(although not specific for osteoclasts) has been used previously toidentify osteoclast-like cells, and correlates, strongly with tartrateresistant acid phosphatase staining. Furthermore, CD51-positive cellsdemonstrate more concentrated staining and are easier to count thancells stained with tartrate resistant acid phosphatase. The sections arethen incubated with biotinylated rabbit antimouse immunoglobulins andABC solution (Vector, Burlingame, Calif.). The sections are washed inphosphate-buffered saline after each antibody step. Positively stainedcells are visualized with diaminobenzidine, 0.01% H₂O₂, 0.3% sodiumazide in 0.05 M Tris buffer pH 7.6. The slides are counterstained withtoluidine blue, dehydrated, and mounted.

Assessment and Statistical Analysis

Histomorphologic analysis is performed of multiple (minimum=5) serial,longitudinal sections stained with hematoxylin and eosin, which enablesthe entire length of the specimen to be visualized on one slide. Thesesections are examined qualitatively using magnifications ranging from40-400× for the cellular and stromal components and the type of bonepresent. A computerized, interactive image analysis system utilizing NIHImage interfaced with a MacIntosh Quadra computer (Apple, Cupertino,Calif.) and a real-time video light microscope system is used to performthe morphometric analysis. One entire section from the center of thespecimen stained with hematoxylin and eosin is used for the morphometricanalysis for bone ingrowth using 100× magnification. The areas formeasurement on the entire section are outlined manually using a mousepad, which was connected to the computer and a television screencontaining the histological image. CD51-positive and negative cells arecounted using 400× magnification using the appropriate sectionsprocessed by immunohistochemistry. All measurements are performedblindly, without knowledge of the treatment.

Histomorphometric analyses are performed to determine total tissue area,total bone area, ratio of bone area divided by total area and expressedas a percentage, and the total number of vitronectin-receptor positivecells abutting the bone and away from the bone in the entire section.

Statistical analysis is accomplished using the StatView software program(Abacus Concepts, Berkeley, Calif.). Parametric statistics are used forassessment of percentage of bone ingrowth, including an analysis ofvariance to compare sequential 3-week treatments. Post hoc tests mayutilize the paired t-test, which compares different treatments performedin the same animals. Cell counting data is analyzed using theKruskal-Wallis test and signed-rank test. A significance level of 0.05is used.

Using the aforementioned procedure, the effects of anti-M-CSF antibodieson bone formation may be readily analyzed. It is expected that aditionof anti-M-CSF antibodies will increase net bone formation and thusprovide a means to enhance osseointegration of, for example, cementlessimplants.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A method of treating a subject suffering from a disease associatedwith periprosthetic bone loss or increased osteoclastic activity andincreased osteoblastic activity comprising administering to the subjecta non-murine antibody that competes with monoclonal antibody RX1 forbinding to M-CSF by more than 75%, wherein said monoclonal antibody RX1comprises the heavy chain and light chain amino acid sequences set forthin SEQ ID NOs: 2 and 4, respectively.
 2. The method of claim 1 whereinthe non-murine antibody specifically binds to the same epitope of M-CSFas said monoclonal antibody RX1, said epitope comprising at least 4contiguous residues of SEQ ID NO: 120 or
 121. 3. The method of claim 1wherein the non-murine antibody is a monoclonal antibody.
 4. The methodof claim 1 wherein the non-murine antibody is a chimeric antibody, ahumanized antibody, a human engineered antibody, a human antibody, or asingle chain antibody.
 5. The method of claim 1 wherein the non-murineantibody retains an affinity Kd with respect to M-CSF of SEQ ID NO: 9 ofat least 10⁻⁸ M or higher.
 6. The method of claim 1 wherein thenon-murine antibody comprises an amino acid sequence 90% identical toSEQ ID NO:
 24. 7. The method of claim 1 wherein the non-murine antibodycomprises SEQ ID NO:
 24. 8. The method of claim 1 wherein the non-murineantibody comprises at least 3 sequences selected from: (a) the groupconsisting of SEQ ID NOs: 18, 21, 24, 29, 32, and 36; or (b) the groupconsisting of SEQ ID NOs: , 21, 24, 32, 36 and QASQSIGTSIH (SEQ ID NO:138).
 9. The method of claim 1 wherein the non-murine antibody comprisesall of (a) SEQ ID NOs: 18, 21, 24, 29, 32, and 36; or (b) SEQ ID NOs:18, 21, 24, 32, 36 and QASQSIGTSIH (SEQ ID NO: 138).
 10. The method ofclaim 8 wherein the non-murine antibody further comprises one or moreCDRs selected from the group consisting of: SEQ ID NOs: 16, 19, 22, 27,30, 34, 17, 20, 23, 28, 31, 35, 18, 21, 25, 29, 32, 37, 18, 21, 26, 29,33, or
 38. 11. The method of claim 10 wherein the non-murine antibodycomprises a CDR in which at least one amino acid within a CDR issubstituted by a corresponding residue of a corresponding CDR of anotheranti-MCSF antibody.
 12. The method of claim 8 wherein the non-murineantibody comprises a constant region of a human antibody sequence andone or more heavy and light chain variable framework regions of a humanantibody sequence.
 13. The method of claim 12 wherein the human antibodysequence is an individual human sequence, a human consensus sequence, anindividual human germline sequence, or a human consensus germlinesequence.
 14. The method of claim 8 wherein the non-murine antibodycomprises a fragment of an IgG1 constant region.
 15. The method of claim14 wherein the non-murine antibody comprises a mutation in the IgG1constant region that reduces antibody-dependent cellular cytotoxicity orcomplement dependent cytotoxicity activity.
 16. The method of claim 8wherein the non-murine antibody comprises a fragment of an IgG4 constantregion.
 17. The method of claim 16 wherein the non-murine antibodycomprises a mutation in the IgG4 constant region that reduces formationof half-antibodies.
 18. The method of claim 1, wherein the non-murineantibody comprises a heavy chain variable region that comprises theamino acid sequenceXVXLXEXGXXXXXXXXXLXLXCXVXDYSITSDYAWNWIXQXXXXXLXWMGYISYSGSTSXNXXLXXXIXIXLXLXXVXXXDXAXYYCASFDYAHAMDYW GXGTXVXVXX, wherein X isany amino acid.
 19. The method of claim 1, wherein the non-murineantibody comprises a light chain variable region that comprises theamino acid sequenceXIXLXQXXXXXXVXXXXXVXFXCXAXQSIGTSIHWYXQXXXXXPXLLIKYASEXXXXIXXXFXGXGXGXXFXLXIXXVXXXDXADYYCQQINSWPTTFGXGTXLXXXXX, wherein X is anyamino acid.
 20. The method of claim 1 wherein the non-murine antibodycomprises any one of the heavy chain variable region sequences set forthin SEQ ID NOS: 41 or
 43. 21. The method of claim 1 wherein thenon-murine antibody comprises any one of the light chain sequences setforth in SEQ ID NOS: 45, 47, 48, 51, 53 or
 136. 22. The method of claim8 wherein the non-murine antibody comprises a variable heavy chain aminoacid sequence which is at least 80% identical to the variable heavychain amino acid sequence set forth in SEQ ID NOs: 41 or
 43. 23. Themethod of claim 8 wherein the non-murine antibody comprises a variablelight chain amino acid sequence which is at least 80% identical to thevariable light chain amino acid sequence set forth in SEQ ID NOs: 45,47, 48, 51, or
 53. 24. The method of claim 1 further comprisingadministering a second therapeutic agent.
 25. The method of claim 1wherein the antibody is administered at a dose between about 0.1 mg/kgto 30 mg/kg body weight.
 26. A kit comprising a therapeuticallyeffective amount of the antibody of claim 1 or 8 or 22, packaged in acontainer, such as a vial or bottle, and further comprising a labelattached to or packaged with the container, the label describing thecontents of the container and providing indications and/or instructionsregarding use of the contents of the container to treat a subjectsuffering from disease associated with increased osteoclastic activityand increased osteoblastic activity.